Methods for modulating angiogenesis by using the anti-angiogenic angiotensin-7 and polynucleotides encoding therefor

ABSTRACT

The present invention provides methods for modulating angiogenesis by administering anti-angiogenic Ang-7 polypeptides to a subject. Methods of modulating angiogenesis by administering an anti-angiogenic ANG-7 nucleic acid are also provided.

BACKGROUND OF THE INVENTION

The cellular behavior responsible for the development, maintenance andrepair of differentiated cells and tissues is regulated, in large part,by intercellular signals conveyed via growth factors and other ligandsand their receptors. The receptors for these intracellular signalingmolecules are located on the cell surface of responding cells. Growthfactors and other ligands bind to the receptors, thereby causingtransduction of a signal across the cell membrane. Such signaltransduction can occur by many modes, including pore formation andphosphorylation. Phosphorylation of tyrosines on proteins by tyrosinekinases is one of the key modes by which signals are transduced acrossthe cell membrane. Indeed, several currently known protein tyrosinekinase genes encode transmembrane receptors for polypeptide growthfactors and hormones.

Angiogenesis is generally thought to be heavily regulated by growthfactors and other ligands. Angiogenesis, and the concurrent tissuedevelopment and regeneration, depends on the tightly controlledprocesses of endothelial cell proliferation, migration, differentiationand survival. Both stimulator and inhibitor ligands appear to interact,directly or indirectly, with cellular receptors during these processes.Angiogenesis begins with the erosion of the basement membrane by enzymesreleased by endothelial cells and leukocytes. The endothelial cells,which line the lumen of blood vessels, then protrude through thebasement membrane. Angiogenic stimulators induce endothelial cells tomigrate through the eroded basement membrane. The migrating cells thenform a “sprout” off the parent blood vessel, where the endothelial cellsundergo mitosis and proliferate. The endothelial sprouts merge with eachother to form capillary loops, creating the new blood vessel.

The ligands and receptors involved in endothelial cell regulation arebeginning to be elucidated. In particular, endothelial growth factorreceptors and their kinases have been discovered. For example, a geneencoding an endothelial cell transmembrane tyrosine kinase was describedby Partanen et al. (Proc. Natl. Acad. Sci. USA 87:8913-17 (1990)). Thisgene and its encoded protein are called “TIE,” which is an abbreviationfor “tyrosine kinase with Ig and EGF homology domains.” (See Partanen etal., Mol. Cell. Biol. 12:1698-1707 (1992); International PatentPublication WO 99/15653.) Enhanced TIE expression was shown duringneovascularization to be associated with developing ovarian folliclesand granulation tissue in skin wounds. (See Korhonen et al., Blood80:2548-2555 (1992).) Thus, TIE protein is likely to play a role inangiogenesis.

Two structurally related rat TIE receptor-like tyrosine kinases, TIE-1and TIE-2, have been reported; these receptors are encoded by distinctgenes. (See Maisonpierre et al., Oncogene 8:1631-7 (1993).) Both geneswere found to be widely expressed in endothelial cells of embryonic andpostnatal tissues. Significant levels of TIE-2 transcripts were alsopresent in other embryonic cell populations, including lens epithelium,heart epicardium and regions of mesenchyme. (See Maisonpierre et. al.,supra.) The predominant expression of TIE receptors in vascularendothelia suggests that TIE plays a role in the development andmaintenance of the vascular system, and in particular, angiogenesis.

Ligands of the TIE receptors have also been characterized. Two TIE-2binding ligands, angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), havebeen identified. Ang-1 polypeptide interacts with the TIE-2 receptortyrosine kinase. (See Maisonpierre et al., Science 277:55-60 (1997).)Ang-2 polypeptide is antagonistic to Ang-1 polypeptide, preventingbinding of the activating ligand and blocking its ability to stimulateTIE-2 kinase activity and autophosphorylation. Ang-1 and Ang-2 do notbind TIE-1, however. Ang-1 and Ang-2 are about 60% identical; the aminoacid sequences of these polypeptides share similar domain structure withan N-terminal coiled-coil region and a C-terminal fibrinogen-likedomain. Northern (RNA) analysis shows that ANG-1 RNA is quite widelyexpressed, but that the expression of ANG-2 RNA is very limited. ANG-2RNA is present only in tissues such as ovary, uterus, and placenta,which undergo vascular remodeling. Ang-1 is thought to be the same asthe human TIE receptor ligand “htie-2” or “hTL-1.” (See InternationalPatent Publication WO 99/15653.)

Recently, other ligands for the TIE-2 receptor were identified. (SeeValenzuela et al., Proc. Natl. Acad. Sci. USA 96:1904-09 (1999).) Theseligands are called TIE ligand-3 (or angiopoietin-3 (Ang-3)) and TIEligand-4 (or angiopoietin-4 (Ang-4)). Ang-3, a mouse polypeptide,appears to be antagonistic to Tie2 receptor while Ang-4, a humanpolypeptide, appears to be an agonist. The precise physiological role ofAng-3 and Ang-4 polypeptides remains to be elucidated.

Other TIE ligand homologues from humans, NL1 to NL6 and NL8, have alsobeen identified. (See International Patent Publications WO 99/15653 andWO 99/15654.) One of these homologues, NL6, was identified by screeninga cDNA library for sequences that encode secretory signals. Subsequentanalysis of the full length NL6 cDNA revealed homology to TIE ligandreceptors. The other homologues, NL1-5 and NL8, were identified byscreening an EST database for sequences showing similarity to NL6. Basedon their similarity to NL6, NL1-5 and NL8, were also proposed to beinvolved in angiogenesis. NL1 and NL8 were found to be capable of makingcells tumorigenic. (See International Patent Publication WO 99/15653.)

The number of TIE ligand homologues, including Ang-1 to Ang-4 and the NLfamily, suggests that these ligands play diverse roles in angiogenesis.Further characterization of these ligands is, therefore an importantstep in understanding their roles in angiogenesis. In particular,persistent, unregulated angiogenesis occurs in a multiplicity of diseasestates, including tumor metastasis and abnormal growth by endothelialcells, and supports the pathological damage seen in these conditions.Thus, characterization of angiogenic factors may also facilitate thedevelopment of treatments for diseases related to (and hypothesized asbeing related to) angiogenesis. For example, tumor formation has beenproposed to be dependent on angiogenesis. Thus, TIE ligand homologuesthat inhibit angiogenesis may provide therapeutic treatments for suchtumors.

SUMMARY OF THE INVENTION

The present invention provides methods for modulating angiogenesis usinganti-angiogenic Ang-7 polypeptides. The present invention furtherencompasses the use of Ang-7 polypeptides for the treatment of a diseaseor clinical condition where angiogenesis is relevant to the causation ortreatment of the disease or clinical condition. In one embodiment, suchdiseases or conditions include, but are not limited to, cancer, woundhealing, tumor formation, diabetic retinopathies, macular degeneration,cardiovascular diseases, and the like. Further uses of the Ang-7polypeptides include treatment of clinical conditions involvingangiogenesis in the reproductive system, including regulation ofplacental vascularization or use as an abortifacient. The presentinvention also encompasses pharmaceutical compositions containing theAng-7 polypeptide and the use of such pharmaceutical compositions forthe treatment of the above-mentioned diseases or clinical conditions.

One aspect of the present invention relates to the use of Ang-7polypeptides having the amino acid sequence of SEQ ID NO:2, as well asbiologically active or diagnostically or therapeutically usefulfragments, variants, derivatives and analogs thereof. An additionalaspect relates to the use of antibodies against the Ang-7 polypeptidesof the present invention, especially antibodies which bind specificallyto an epitope of the sequence described in SEQ ID NO:2, or a sequencethat shares at least 60%, preferably at least 70%, more preferably atleast 80%, still more preferably at least 90%, or most preferably atleast 95% sequence identity over at least 20, preferably at least 30,more preferably at least 40, still more preferably at least 50, or mostpreferably at least 100 residues, to SEQ ID NO:2.

Another aspect of the present invention relates to the use of isolatedANG-7 nucleic acids encoding the Ang-7 polypeptides of the presentinvention, including mRNAs, DNAs, cDNAs, genomic DNA, as well as ANG-7antisense nucleic acids. Such nucleic acids include the ANG-7 cDNAsequence having the nucleotide sequence of SEQ ID NO: 1. Another aspectrelates to ANG-7 sequence fragments or variants that encode biologicallyactive or diagnostically or therapeutically useful polypeptides. Suchfragments or variants include sequences having all possible codonchoices for the same amino acid or conservative amino acid substitutionsthereof, such as the nucleotide sequence identified as NL1 inInternational Patent Publication WO 99/15653 (SEQ ID NO: 1), thedisclosure of which is incorporated in its entirety by reference herein.Other variants include those nucleic acids that are capable ofselectively hybridizing to a human ANG-7 cDNA (e.g., SEQ ID NO:1) understringent hybridization conditions. Another aspect of the presentinvention relates to nucleic acid probes comprising polynucleotides ofsufficient length to selectively hybridize to a polynucleotide encodingan Ang-7 polypeptide of the present invention.

Still another aspect of the present invention relates to processes forproducing Ang-7 polypeptides, or biologically active and diagnosticallyor therapeutically useful fragments or variants thereof, by recombinanttechniques through the use of recombinant vectors. A further aspect ofthe present invention relates to recombinant prokaryotic and/oreukaryotic host cells comprising an ANG-7 nucleic acid sequence encodingan Ang-7 polypeptide, or biologically active or diagnostically ortherapeutically useful fragments or variants thereof. In a relatedaspect, nucleic acid constructs are provided that express ANG-7 nucleicacids and/or Ang-7 polypeptides, fragments or variants. Such constructstypically include a transcriptional promoter and a transcriptionalterminator, each operably linked for expression of the ANG-7 nucleicacid or fragment thereof.

Another aspect of the present invention relates to processes involvingexpression of the polypeptides, or polynucleotides encoding thepolypeptides, of the present invention for purposes of gene therapy. Asused herein, gene therapy is defined as the process of providing for theexpression of nucleic acid sequences of exogenous origin in anindividual for the treatment of a disease condition within thatindividual.

A further aspect of the present invention relates to processes forutilizing Ang-7 polypeptides fragments, variants, derivatives, oranalogs thereof, or ANG-7 polynucleotides or fragments, variants orderivatives thereof, for therapeutic purposes involving the modulationof angiogenesis, or the modulation of diseases or conditions in whichangiogenesis is relevant to the disease or condition. Such diseases orconditions include, for example, the treatment of cancer, wound healing,diabetic retinopathies, macular degeneration, cardiovascular diseases,and clinical conditions involving angiogenesis in the reproductivesystem, including regulation of placental vascularization or use as anabortifacient. Such treatments further include the use of the Ang-7polypeptides in protein replacement therapy and protein mimetics.

Another aspect of the present invention relates to diagnostic assays fordetecting diseases or clinical conditions, or the susceptibility todiseases or clinical conditions, related to mutations in an ANG-7nucleic acid sequence of the present invention and for detectingover-expression or underexpression of Ang-7 polypeptides encoded by suchsequences.

These and other aspects of the invention will become evident uponreference to the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence of ANG-7 cDNA.

FIG. 2 depicts the amino-acid sequence of human Ang-7 polypeptide. Thesequence is shown in the one letter code of amino-acids.

FIG. 3 depicts an alignment of the Ang-7 (SEQ ID NO:2) amino acidsequence with those of Ang-1 (SEQ ID NO:3), Ang-2 (SEQ ID NO:4), Ang-3(SEQ ID NO:5), and Ang-4 (SEQ ID NO:6). Identical amino acids areindicated by boxes.

FIG. 4 depicts an expression profile of ANG-7 RNA in human tissues. Theexpression levels of ANG-7 RNA in different tissues is shown. Along thehorizontal axis, the reference numerals identify the following tissues:

1-whole brain 28-interventricular 56-liver 2-cerebral cortex septum57-pancreas 3-frontal lobe 29-apex of the heart 58-adrenal gland4-parietal lobe 30-esophagus 59-thyroid gland 5-occipital lobe31-stomach 60-salivary gland 6-temporal lobe 32-duodenum 61-mammarygland 7-paracentral gyrus of 33-jejunum 62-Leukemia HL-60 cerebralcomplex 34-ileum 63-HeLa S3 8-pons 35-ilocecum 64-Leukemia K-5629-cerebellum left 36-appendix 65-Leukemia MOLT-4 10-cerebellum right37-colon ascending 66-Burkitt's lymphoma, 11-corpus callusum 38-colontransverse Raji 12-amygdala 39-rectum 67-Burkitt's 13-caudate nucleus40-kidney lymphoma, 14-hippocalamus 41-skeletal muscle Daudi 15-medullaoblongata 42-spleen 68-colorect. adenocarc. 16-putamen 43-thymus SW-48017-subtantia nigra 44-peripheral blood 69-Lung carcinoma 18-accumbensnucleus 45-lymph node A549 19-thalamus 46-bone marrow 70-fetal brain20-pituitary gland 47-trachea 71-fetal heart 21-spinal cord 48-lung72-fetal kidney 22-heart 50-placenta 73-fetal liver 23-aorta 51-bladder74-fetal spleen 24-atrium left 52-uterus 75-fetal thymus 25-atrium right53-prostate 76-fetal lung 26-ventricle left 54-testis 27-ventricle right55-ovary

FIG. 5 depicts the results of the in vitro translation of Ang-7 RNA.Lane 1: Rainbow [¹⁴C]methylated protein molecular weight markers(Amersham, Little Chalfont Buckinghamshire, England) containing thefollowing proteins: ovalbumin (46 kDa), carbonic anhydrase (30 kDa),tsypsin inhibitor (21.5 kDa), lysozyme (14.3 kDa), and aprotinin (6.5kDa). Lane 2: In vitro translation products of ANG-7 RNA using the T7promoter of the mammalian expression vector pcDNA3.1/Myc-His(−)(Invitrogen, Groningen, Netherlands). Lane 3: In vitro translationproducts of RNA using the SP6 promoter of the mammalian expressionvector pcDNA3.1/Myc-His(−) (negative control). Lane 4: Positive controlfrom the in vitro translation system (Promega, Madison, USA).

FIG. 6 depicts Western blot analysis of the cell extract of CHO cellstransiently transfected with an ANG-7 expression construct. Lane 1:protein molecular weight markers. Lane 2: CHO cells (negative control).Lane 3: transiently transfected CHO cells expressing Ang-7 polypeptide.The Ang-7 polypeptide band is marked by an arrow.

FIG. 7 depicts a Western blot analysis of Ang-7 polypeptide in the celllysate of a stably transfected HEK293 cell clone. Lane 1: proteinmolecular weight markers. Lane 2: cell lysate from HEK293 cells(negative control). Lane 3: cell lysate from stably transfected HEK293cells expressing Ang-7 polypeptide.

FIG. 8 depicts a Western blot analysis of conditioned media from astably transfected HEK293 cell clone expressing Ang-7 polypeptide. Lane1: conditioned media from HEK293 cells (negative control). Lane 2:conditioned media from the stably transfected HEK293 cell cloneexpressing Ang-7 polypeptide.

FIGS. 9A and 9B depict the results of purification of Ang-7 polypeptidefrom conditioned media. FIG. 9A: Western blot analysis; FIG. 9B:coomassie-stained SDS gel of purified Ang-7 polypeptide. The arrows inFIG. 9B indicate the position of recombinant Ang-7 polypeptide. For eachfigure, Lanes 1-5 are fractions 1-5 eluted from the Ni-NTA agarosecolumn. Lane “neg. C” indicates the negative control (conditioned mediafrom HEK293 cells). In FIG. 9B, the double bands probably representdifferent glycosylation forms of Ang-7 polypeptide.

DETAILED DESCRIPTION

Prior to setting forth the invention in more detail, it may be helpfulto a further understanding thereof to set forth definitions of certainterms as used hereinafter.

Definitions:

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Other methods and materialssimilar to those described herein can be used in the practice or testingof the present invention; thus, only exemplary methods and materials aredescribed. For purposes of the present invention, the following termsare defined below.

The term “angiogenesis” means the generation of new blood vessels in atissue or organ. Angiogenesis includes neovascularization and collateralvascularization. “Normal angiogenesis” includes, under normalphysiological conditions, new blood vessel formation associated withwound healing, fetal and embryonal development and formation of thecorpus luteum, endometrium and placenta. “Unwanted angiogenesis” refersto angiogenesis that occurs under abnormal physiological conditions,such as in a disease or clinical condition associated with pathologicaldamage related to the uncontrolled angiogenesis. For example, unwantedangiogenesis can occur during tumor formation, where neovascularizationis present within the tumor.

The term “ANG-7 nucleic acids” (ie., in all caps and italicized) refersto polynucleotides encoding Ang-7 polypeptides, including mRNAs, DNAs,cDNAs, genomic DNA, as well as antisense nucleic acids, andpolynucleotides encoding biologically active or diagnostically ortherapeutically useful fragments, variants and derivatives thereof.Useful fragments and variants include those based on all possible codonchoices for the same amino acid, and codon choices based on conservativeamino acid substitutions, and biologically active or diagnostically ortherapeutically useful fragments, or derivatives thereof. Usefulvariants further include those having at least 70% polynucleotidesequence identity, more preferably 80%, still preferably 90%, to thepolynucleotide of SEQ ID NO:1, and biologically active anddiagnostically or therapeutically useful fragments, or derivativesthereof.

The term “ANG-7 gene” (ie, in all caps and italicized) refers to codingsequences, intervening sequences and regulatory elements controllingtranscription and/or translation.

The terms “polynucleotide” and “nucleic acid” refer to a polymercomposed of a multiplicity of nucleotide units (ribonucleotide ordeoxyribonucleotide or related structural variants), which are typicallylinked via phosphodiester bonds. A polynucleotide or nucleic acid can beof substantially any length, typically from about six (6) nucleotides toabout 10⁹ nucleotides or larger. Polynucleotides or nucleic acidsinclude RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers,both sense and antisense strands, and can also be chemically orbiochemically modified or can contain non-natural or derivatizednucleotide bases, as will be readily appreciated by the skilled artisan.Such modifications include, for example, labels, methylation,substitution of one or more of the naturally occurring nucleotides withan analog, internucleotide modifications such as uncharged linkages(e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, and the like), charged linkages (e.g., phosphorothioates,phosphorodithioates, and the like), pendent moieties (e.g.,polypeptides), intercalators (e.g., acridine, psoralen, and the like),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, and the like). Also included are synthetic molecules thatmimic polynucleotides in their ability to bind to a designatednucleotide sequence via hydrogen bonding and other chemicalinteractions. Such molecules are known in the art and include, forexample, those in which peptide linkages substitute for phosphatelinkages in the backbone of the molecule.

The term “oligonucleotide” refers to a polynucleotide of from about six(6) to about one hundred (I 00) nucleotides, or more in length. Thus,oligonucleotides are a subset of polynucleotides. Oligonucleotides canbe synthesized on an automated oligonucleotide synthesizer (e.g., thosemanufactured by Applied BioSystems (Foster City, Calif.)), according tospecifications provided by the manufacturer.

The term “primer” refers to a polynucleotide, typically anoligonucleotide, whether occurring naturally, as in an enzyme digest, orproduced synthetically in vitro, which acts as a point of initiation ofpolynucleotide synthesis when used under conditions in which a primerextension product is synthesized.

The term “Ang-7 polypeptide” refers to polypeptides having the aminoacid sequence of SEQ ID NO:2, and biologically active or diagnosticallyor therapeutically useful fragments, variants and derivatives thereof.“Fragment” refers to a portion of an Ang-7 polypeptide having typicallyat least 10 contiguous amino acids, more typically at least 20, stillmore typically at least 50 contiguous amino acids of the Ang-7polypeptide. Useful variants typically include those having conservativeamino acid substitutions, and biologically active and diagnostically ortherapeutically useful fragments thereof. Useful variants are typicallyat least about 50% similar to the native Ang-7 amino acid sequence (SEQID NO:2), more typically in excess of about 90%, and still moretypically at least about 95% similar, and biologically active, ordiagnostically or therapeutically useful fragments or derivativesthereof. Ang-7 polypeptides further include those that areimmunologically cross-reactive with anti-Ang-7 polypeptides

The term “polypeptide” refers to a polymer of amino acids and itsequivalent and does not refer to a specific length of the product; thus,peptides and oligopeptides (i.e., fragments) and proteins are includedwithin the definition of a polypeptide. This term also includesderivatives of the Ang-7 polypeptide, for example, glycosylations,acetylations, phosphorylations, and the like. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (e.g., unnatural amino acids, and the like),polypeptides with substituted linkages as well as other modificationsknown in the art, both naturally and non-naturally occurring.

The term “biologically active” refers to the ability of a molecule tomodulate angiogenesis, such as by affecting endothelial tube formation(e.g., using the HUVEC assay of Example 9 (infra)), or that affectstumor cell growth or proliferation (e.g., using the tumor cell growthinhibition assay of Example 10 (infra)). Biologically active moleculescan be Ang-7 polypeptides, fragments, variants, derivatives and analogsthereof; nucleic acids encoding Ang-7 polypeptides, fragments, variantsand derivatives thereof; and anti-Ang-7 antibodies, which modulateangiogenesis (e.g., inhibiting or stimulating endothelial tubeformation) or by modulating tumor cell growth or proliferation (e.g.,inhibiting or stimulating tumor cell growth).

The terms “therapeutically useful” or “therapeutically effective” referto an amount of a molecule (e.g., an Ang-7 polypeptide, anti-Ang-7antibody, or ANG-7 nucleic acid) that is sufficient to modulateangiogenesis (e.g. inhibiting or stimulating endothelial tube formation)or to modulate tumor cell growth or proliferation (e.g., inhibiting orstimulating tumor cell growth) in a subject, such as a patient or amammal.

The terms “diagnostically useful” or “diagnostically effective” refer toa molecule (e.g., an Ang-7 polypeptide, anti-Ang-7 antibody, or ANG-7nucleic acid) for detecting angiogenesis, or the inhibition ofangiogenesis, in a subject. These terms further include molecules usefulfor detecting diseases or clinical conditions, or the susceptibility todiseases or clinical conditions, related to mutations in an ANG-7nucleic acid sequence of the present invention and for detectingover-expression or underexpression of Ang-7 polypeptides encoded by suchsequences.

The term “Ang-7 compounds” or “Ang-7 anti-angiogenic compounds” refersto biologically active Ang-7 polypeptide, fragments, variants,derivatives, or analogs thereof, to anti-Ang-7 antibodies, tobiologically active ANG-7 nucleic acids, fragments or derivatives, andto ANG-7 antisense nucleic acids.

The terms “amino acid,” “amino acid residue,” or “residue” refer tonaturally occurring L amino acids or to D amino acids as describedfurther below. Amino acids are referred to herein by either theircommonly known three letter symbols or by the one-letter symbolsrecommended by the IUPAC-IUB Biochemical Nomenclature Commission.Nucleotides, likewise, may be referred to by their commonly acceptedsingle-letter codes. (See, e.g., Bruce Alberts et al., Molecular Biologyof the Cell, Garland Publishing, Inc., New York (3d ed. 1994)).

The terms “identical” or “percent identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acids that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms, or by visualinspection.

The term “substantially identical,” in the context of two nucleic acids,or two polypeptide sequences, refers to two or more sequences orsubsequences that have at least 60%, typically 80%, most typically90-95% identity, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow, or by visual inspection. An indication that two polypeptidesequences are “substantially identical” is that one polypeptide isimmunologically reactive with antibodies raised against the secondpolypeptide.

“Similarity” or “percent similarity” in the context of two orpolypeptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesor conservative substitutions thereof, that are the same, when comparedand aligned for maximum correspondence, as measured using one of thefollowing sequence comparison algorithms, or by visual inspection. Byway of example, a first protein region can be considered similar to aregion of human Ang-7 polypeptide when the amino acid sequence of thefirst region is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or even95% identical, or conservatively substituted, to a region of Ang-7polypeptide when compared to any sequence in Ang-7 polypeptide of anequal number of amino acids as the number contained in the first region,or when compared to an aligned sequence of Ang-7 polypeptide that hasbeen aligned by a computer similarity program known in the art, asdiscussed above.

The term “substantial similarity” in the context of polypeptidesequences, indicates that the polypeptide comprises a sequence with atleast 70% sequence identity to a reference sequence, or preferably 80%,or more preferably 85% sequence identity to the reference sequence, ormost preferably 90% identity over a comparison window of about 10-20amino acid residues. In the context of amino acid sequences,“substantial similarity” further includes conservative substitutions ofamino acids. Thus, a polypeptide is substantially similar to a secondpolypeptide, for example, where the two peptides differ only by one ormore conservative substitutions.

The term “conservative substitution,” when describing a polypeptide,refers to a change in the amino acid composition of the polypeptide thatdoes not substantially alter the polypeptide's activity. Thus, a“conservative substitution” of a particular amino acid sequence refersto amino acid substitutions of those amino acids that are not criticalfor protein activity or substitution of amino acids with other aminoacids having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or non-polar, etc.) such that thesubstitutions of even critical amino acids do not substantially alteractivity. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following six groupseach contain amino acids that are conservative substitutions for oneanother:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

(See also Creighton, Proteins, W. H. Freeman and Company (1984).) Inaddition, individual substitutions, deletions or additions that alter,add or delete a single amino acid or a small percentage of amino acidsin an encoded sequence are also “conservative substitutions.”

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, coordinates are designated, if necessary, and sequencealgorithm program parameters are designated. The sequence comparisonalgorithm then calculates the percent sequence identity for the testsequence(s) relative to the reference sequence, based on the designatedprogram parameters.

Optimal alignment of sequences for comparison can be conducted, forexample, by the local homology algorithm of Smith & Waterman (Adv. Appl.Math. 2:482 (1981)), by the homology alignment algorithm of Needleman &Wunsch (J. Mol. Biol. 48:443 (1970)), by the search for similaritymethod of Pearson & Lipman (Proc. Natl. Acad. Sci. USA 85:2444 (1988)),by computerized implementations of these algorithms (g., GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Dr., Madison, Wis.), or by visualinspection. (See generally Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, New York (1996).)

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show the percent sequence identity. It also plotsa tree or dendogram showing the clustering relationships used to createthe alignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle (J. Mol. Evol. 35:351-360 (1987)). The methodused is similar to the method described by Higgins & Sharp (CABIOS5:151-153 (1989)). The program can align up to 300 sequences, each of amaximum length of 5,000 nucleotides or amino acids. The multiplealignment procedure begins with the pairwise alignment of the two mostsimilar sequences, producing a cluster of two aligned sequences. Thiscluster is then aligned to the next most related sequence or cluster ofaligned sequences. Two clusters of sequences are aligned by a simpleextension of the pairwise alignment of two individual sequences. Thefinal alignment is achieved by a series of progressive, pairwisealignments. The program is run by designating specific sequences andtheir amino acid or nucleotide coordinates for regions of sequencecomparison and by designating the program parameters. For example, areference sequence can be compared to other test sequences to determinethe percent sequence identity relationship using the followingparameters: default gap weight (3.00), default gap length weight (0.10),and weighted end gaps. Another useful program for the multiple alignmentof sequences is MEGALIGN™ Expert Sequence Analysis Software (DNASTAR,Madison, Wis.).

Another example of an algorithm that is suitable for determining percentsequence identity and similarity is the BLAST algorithm, which isdescribed by Altschul et al. (J. Mol. Biol. 215:403-410 (1990)). (Seealso Zhang et al., Nucleic Acid Res. 26:3986-90 (1998); Altschul et al.,Nucleic Acid Res. 25:3389-402 (1997). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al. (1990),supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extension of the word hitsin each direction is halted when: the cumulative alignment score fallsoff by the quantity X from its maximum achieved value; the cumulativescore goes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (SeeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9 (1992)),alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA90:5873-77 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more typically less than about0.01, and most typically less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two polypeptides differ only byconservative substitutions.

The terms “transformation” or “transfection” means the process of stablyaltering the genotype of a recipient cell or microorganism by theintroduction of polynucleotides. This is typically detected by a changein the phenotype of the recipient cell or organism. The term“transformation” is generally applied to microorganisms, while“transfection” is used to describe this process in cells derived frommulticellular organisms.

Methodologies for polymerase chain reaction (“PCR”) are generallydisclosed in U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,800,159. Othernomenclature used herein and many of the laboratory procedures in cellculture, molecular genetics and nucleic acid chemistry andhybridization, which are described below, are those well known andcommonly employed in the art. (See generally Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, New York (1996);Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, New York (1989)). Standardtechniques are used for recombinant nucleic acid methods, polynucleotidesynthesis, preparation of biological samples, preparation of cDNAfragments, isolation of mRNA and the like. Generally enzymatic reactionsand purification steps are performed according to the manufacturers'specifications.

The present invention provides methods for modulating angiogenesis usinganti-angiogenic Ang-7 polypeptides and biologically active ordiagnostically or therapeutically useful fragments, variants,derivatives or analogs thereof. The present invention furtherencompasses methods of using ANG-7 nucleic acids, as more fullydescribed below, to modulate angiogenesis. The present invention furtherencompasses the use of Ang-7 polypeptides and/or ANG-7 nucleic acids forthe treatment of a disease or clinical condition where angiogenesis isrelevant to the causation or treatment of the disease or clinicalcondition, including but not limited to cancer, wound healing, tumorformation, diabetic retinopathies, macular degeneration, cardiovasculardiseases, and the like.

ANG-7 Nucleic Acids:

One aspect of the present invention relates to isolated nucleic acidsencoding Ang-7 polypeptides, including mRNAs, DNAs, cDNAs, genomic DNA,as well as antisense nucleic acids, and their use in modulatingangiogenesis. ANG-7 nucleic acids include the ANG-7 cDNA sequence (e.g.,SEQ ID NO: 1). ANG-7 nucleic acids further include biologically activesequence variants, such as those encoding all possible codon choices forthe same amino acid or conservative amino acid substitutions thereof,and also include diagnostically or therapeutically useful fragmentsthereof. Such variants include the NL1 cDNA sequence (SEQ ID NO: 1 ofInternational Patent Publication WO 99/15653).

The invention further provides purified ANG-7 nucleic acids comprisingat least 6 contiguous nucleotides (e.g., a hybridizable portion)encoding a fragment of an Ang-7 polypeptide. In another embodiment, theANG-7 nucleic acids consist of fragments of at least 8 (contiguous)nucleotides, 25 nucleotides, 50 nucleotides, 100 nucleotides, 150nucleotides, 200 nucleotides, or even up to 250 nucleotides or more ofan ANG-7 sequence. In another embodiment, the nucleic acids are largerthan 200 or 250 nucleotides in length. The nucleic acids can be single-or double-stranded. As is readily apparent, as used herein, a “nucleicacid encoding a fragment of an Ang-7 polypeptide” is construed asreferring to a nucleic acid encoding only the recited fragment orportion of the Ang-7 polypeptide and not the other contiguous portionsof the Ang-7 polypeptide as a continuous sequence. Fragments of ANG-7nucleic acids encoding one or more Ang-7 domains are provided.

The invention also relates to nucleic acids hybridizable to, orcomplementary to, the foregoing sequences, Such nucleic acids includemRNAs, DNAs, cDNAs, genomic DNA, as well as antisense nucleic acids, andbiologically active and diagnostically or therapeutically usefulfragments or variants thereof. Nucleic acids are also provided whichcomprise a sequence complementary to at least 10, 25, 50, 100, 200, or250 nucleotides or more of an ANG-7 gene or cDNA. In one embodiment, anucleic acid is hybridizable to an ANG-7 nucleic acid (e.g., havingsequence SEQ ID NO: 1), or to a nucleic acid encoding an ANG-7 variant,under conditions of high stringency is provided.

By way of example, and not limitation, procedures using conditions ofhigh stringency are as follows: Prehybridization of filters containingDNA is carried out for 8 hours to overnight at 65° C. in buffer composedof 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 (μg/ml denatured salmon sperm DNA). Filters arehybridized for 48 hours at 65° C. in prehybridization mixture containing100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeledprobe. Washing of filters is done at 37° C. for 1 hour in a solutioncontaining 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This isfollowed by a wash in 0.1×SSC at 50° C. for 45 min beforeautoradiography. Other conditions of high stringency, which can be used,are well known in the art. (See generally Ausubel et al., supra).

In another embodiment, a nucleic acid which is hybridizable to an ANG-7nucleic acid under conditions of moderate stringency is provided. By wayof example, and not limitation, procedures using such conditions ofmoderate stringency are as follows: Prehybridization of filterscontaining DNA is carried out for 8 hours to overnight at 55° C. inbuffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP,0.2% Ficoll, 0.02% BSA and 500 μg/ml denatured salmon sperm DNA. Filtersare hybridized for 24 hours at 55° C. in a prehybridization mixturecontaining 100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of³²P-labeled probe. Washing of filters is done at 37° C. for 1 hour in asolution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA.

In another embodiment, a nucleic acid which is hybridizable to an ANG-7nucleic acid under conditions of low stringency is provided. By way ofexample, and not limitation, procedures using such conditions of lowstringency are as follows: Filters containing DNA are pretreated for 6hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mMTris-HCl (pH 7.5), 5 mM EDTA, 0.1% polyvinylpyrrolidone (PVP), 0.1%Ficoll, 1% bovine serum albumin (BSA), and 500 μg/ml denatured salmonsperm DNA. Hybridizations are carried out in the same solution with thefollowing modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/mlsalmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×10⁶ cpm³²P-labeled probe. Filters are incubated in hybridization mixture for18-20 hours at 40° C., and then washed for 1.5 hours at 55° C. in asolution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1%SDS. The wash solution is replaced with fresh solution and incubated anadditional 1.5 hours. Filters are blotted dry and exposed forautoradiography. Other conditions of low stringency that can be used arewell known in the art (those employed for cross-species hybridizations).(See also Shilo and Weinberg, Proc. Natl. Acad. Sci. USA 78:6789-6792(1981)).

Low, moderate and high stringency conditions are well known to those ofskill in the art, and will vary predictably depending on the basecomposition of the particular nucleic acid sequence and on the specificorganism from which the nucleic acid sequence is derived. For guidanceregarding such conditions see, for example, Sambrook et al., MolecularCloning, A Laboratory Manual (Second Edition, Cold Spring Harbor Press,NY, pp. 9.47-9.57 (1989)); and Ausubel et al., Current Protocols inMolecular Biology (Green Publishing Associates and Wiley Interscience,NY (1989)).

Specific embodiments for the cloning of an ANG-7 nucleic acid, presentedas a particular example but not by way of limitation, are as follows.

For expression cloning (a technique commonly known in the art), anexpression library is constructed by methods known in the art. Forexample, mRNA (e.g., human) is isolated, cDNA is prepared and thenligated into an expression vector (e.g., a bacteriophage derivative)such that it is capable of being expressed by the host cell into whichit is then introduced. Various screening assays can then be used toselect for the expressed Ang-7 polypeptide. In one embodiment,anti-Ang-7 specific antibodies can be used for selection.

In another embodiment, polymerase chain reaction (PCR) is used toamplify the desired sequence in a genomic or cDNA library prior toselection. Oligonucleotides representing known ANG-7 sequences, forexample, as selected from SEQ ID NO: 1, can be used as primers in PCR.In a typical embodiment, the oligonucleotide represents at least part ofthe ANG-7 conserved segments of sequence identity between ANG-7 ofdifferent species. The synthetic oligonucleotides can be utilized asprimers to amplify particular sequences within an ANG-7 gene by PCRusing nucleic acids from a source (RNA or DNA), typically a cDNAlibrary, of potential interest. PCR can be carried out, for example, byuse of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (GeneAmp). The DNA being amplified can include mRNA or cDNA or genomic DNAfrom any eukaryotic species. One of skill in the art can choose tosynthesize several different degenerate primers for use in the PCRreactions.

It is also possible to vary the stringency of hybridization conditionsused in priming the PCR reactions, to allow for greater or lesserdegrees of nucleotide sequence similarity between the known ANG-7sequence and the related nucleic acid being isolated. For cross specieshybridization, low stringency conditions are typically used. For samespecies hybridization, moderately stringent conditions are moretypically used. After successful amplification of a segment of a relatedANG-7 nucleic acid, that segment can be molecularly cloned andsequenced, and utilized as a probe to isolate a complete cDNA or genomicclone. This, in turn, can permit the determination of a completenucleotide sequence, the analysis of its expression, and the productionof its protein product for functional analysis, as described infra. Inthis fashion, additional nucleic acids encoding Ang-7 polypeptides andAng-7 polypeptide variants can be identified.

The above-methods are not meant to limit the following generaldescription of methods by which ANG-7 nucleic acids can be obtained. Anyeukaryotic cell potentially can serve as the nucleic acid source for themolecular cloning of ANG-7 nucleic acids. The nucleic acids encodingAng-7 polypeptide can be isolated from vertebrate sources including,mammalian sources such as, porcine, bovine, feline, avian, equine,canine and human as well as additional primate sources. The DNA can beobtained by standard procedures known in the art from cloned DNA (e.g.,a DNA “library”), by chemical synthesis, by cDNA cloning, or by thecloning of genomic DNA, or fragments thereof, purified from the desiredcell. (See e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989); Glover, (ed.), DNA Cloning: A Practical Approach,MRL Press, Ltd., Oxford, U.K. Vol. I, II. (1985)). Clones derived fromgenomic DNA can contain regulatory and intron DNA regions in addition tocoding regions; clones derived from cDNA will typically contain onlyexon sequences. Whatever the source, the nucleic acid can be molecularlycloned into a suitable vector for propagation of the nucleic acid.

In the molecular cloning of ANG-7 nucleic acids from genomic DNA, DNAfragments are generated, some of which will encode an ANG-7 gene. TheDNA can be cleaved at specific sites using various restriction enzymes.Alternatively, one can use DNase in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, such as, forexample, by sonication. The linear DNA fragments can then be separatedaccording to size by standard techniques, including but not limited to,agarose and polyacrylamide gel electrophoresis and columnchromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired gene can be accomplished in a number ofways. For example, a portion of an ANG-7 gene, cDNA (of any species) orits specific RNA, or a fragment thereof, can be purified and labeled,the generated DNA fragments can be screened by nucleic acidhybridization to the labeled probe (see, e.g., Benton and Davis, Science196:180 (1975); Grunstein and Hogness, Proc. Natl. Acad. Sci. USA72:3961 (1975)). Those DNA fragments with substantial identity to theprobe will hybridize. It is also possible to identify the appropriatefragment by restriction enzyme digestion(s) and comparison of fragmentsizes with those expected according to a known restriction map, ifavailable. Further selection can be carried out on the basis of theproperties of the gene.

Alternatively, the presence of the gene can be detected by assays basedon the physical, chemical, or immunological properties of its expressedproduct. For example, cDNA clones, or DNA clones which hybrid-select theproper mRNAs, can be selected which produce a polypeptide that, forexample, has similar or identical electrophoretic migration, isoelectricfocusing behavior, proteolytic digestion maps, modulation ofangiogenesis, receptor binding activity, or antigenic properties asknown for Ang-7 polypeptide. Immune serum or an antibody whichspecifically binds to the Ang-7 polypeptide can be used to identifyputatively Ang-7 polypeptide synthesizing clones by binding in an ELISA(enzyme-linked immunosorbent assay)-type procedure.

ANG-7 nucleic acids can also be identified by mRNA selection by nucleicacid hybridization followed by in vitro translation. In this procedure,fragments are used to isolate complementary mRNAs by hybridization. SuchDNA fragments typically represent available, purified DNA of anotherspecies (e.g., human, mouse, and the like). Immunoprecipitation analysesor functional assays (e.g., inhibition of angiogenesis, endothelial tubeformation in vitro tumor inhibition, or binding to a TIE receptor) ofthe in vitro translation products of the isolated mRNAs identifies themRNA and, therefore, the complementary DNA fragments that contain thedesired sequences. In addition, specific mRNAs can be selected byadsorption of polysomes isolated from cells to immunobilized antibodiesspecifically directed against Ang-7 polypeptide. A radiolabeled ANG-7cDNA can be synthesized using the selected mRNA (from the adsorbedpolysomes) as a template. The radiolabeled mRNA or cDNA can then be usedas a probe to identify the ANG-7 nucleic acid fragments from among othergenomic DNA fragments.

Alternatives to isolating the ANG-7 genomic DNA include, but are notlimited to, chemically synthesizing the gene sequence itself from aknown sequence or making cDNA to mRNA that encodes an Ang-7 polypeptide.For example, RNA for cDNA cloning of the ANG-7 gene can be isolated fromcells that express the Ang-7 polypeptide. Other methods are possible.

The identified and isolated ANG-7 nucleic acids can then be insertedinto an appropriate cloning vector. A large number of vector-hostsystems known in the art can be used. Possible vectors include, but arenot limited to, plasmids or modified viruses. The vector system isselected to be compatible with the host cell. Such vectors include, butare not limited to, bacteriophages such as lambda derivatives, yeastintegrative and centromeric vectors, 21 plasmid, and derivativesthereof, or plasmids such as pBR322, pUC or pRSETC (InVitrogen, SanDiego, Calif.) plasmid derivatives or the Bluescript vector (Stratagene,La Jolla, Calif.), to name a few. The insertion of the ANG-7 nucleicacids into a cloning vector can be accomplished, for example, byligating the DNA fragment into a cloning vector which has complementarycohesive termini. If the complementary restriction sites used tofragment the DNA are not present in the cloning vector, however, theends of the DNA molecules can be enzymatically modified. Alternatively,any site desired can be produced by ligating nucleotide sequences(linkers) onto the DNA termini; these ligated linkers can comprisespecific chemically synthesized oligonucleotides encoding restrictionendonuclease recognition sequences. A restriction site can also beintroduced into a nucleic acid by PCR amplification of the nucleic acidusing a primer(s) that encodes the desired restriction site(s). In analternative method, the cleaved vector and ANG-7 nucleic acids can bemodified by homopolymeric tailing. Recombinant molecules can beintroduced into host cells via transformation, transfection, infection,electroporation, and the like, so that many copies of the gene sequenceare generated.

In another method, the ANG-7 gene can be identified and isolated afterinsertion into a suitable cloning vector in a “shot gun” approach.Enrichment for the ANG-7 gene, for example, by size fractionation, canbe done before insertion into the cloning vector. In specificembodiments, transformation of host cells with recombinant DNA moleculesthat incorporate the isolated ANG-7 gene, cDNA, or synthesized DNAsequence enables generation of multiple copies of the gene. Thus, thegene can be obtained in large quantities by growing transformants,isolating the recombinant DNA molecules from the transformants and, whennecessary, retrieving the inserted gene from the isolated recombinantDNA.

In this specific case, the ANG-7 cDNA was isolated by homology withanother known angiopoietin, Ang-1. Briefly, fragments of ANG-7 cDNA wereisolated by a BLAST search (Altschul et al., Nucleic Acids Res.25:3389-402 (1997)) against the Expressed Sequence Tag (“EST”) database(National Center for Biotechnology Information) using the deduced aminoacid sequence of Ang-1 as a reference. Two EST's with overlappingsequences were isolated. A full length cDNA fragment encoding the Ang-7polypeptide was cloned by screening a full-length human cDNA libraryusing a radioactively-labeled EST. Subsequent analysis of the humanANG-7 cDNA (SEQ ID NO:1) identified an open reading frame of 493 aminoacids (SEQ ID NO:2). The deduced amino acid sequence encodes apolypeptide of about 57 kDa. Subsequent analysis of the ANG-7 cDNAconfirms that it directs the synthesis of a recombinantly expressedpolypeptide of that apparent molecular weight, as determined by SDSPAGE. The human Ang-7 polypeptide is 23.9% and 23.5% similar to theAng-1 and -2 polypeptides, respectively. Amino acid conservation isdistributed throughout the length of the two proteins. The expressionprofile of Ang-7 polypeptide indicates that the polypeptide is expressedin a variety of heavily vascularized tissues, including heart tissues,the uterus, mammary gland and corpus callosum.

A clone harboring the ANG-7 cDNA, ANG-7-cDNA/pGEM, was deposited at theDSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Mascheroder Weg 1b, D-38124 Braunschweig, Germany, on Jun. 26, 2000,under Deposit Registration No. DSM 13562. This deposit was made underthe provisions of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purpose of Patent Procedure andthe Regulation thereunder (Budapest Treaty).Ang-7 Polypeptides,Fragments, Variants, Derivatives and Analogs:

The invention further relates to Ang-7 polypeptides, and biologicallyactive or diagnostically or therapeutically useful fragments, variants,derivatives and analogs thereof, and their use in modulatingangiogenesis. In one embodiment, the Ang-7 polypeptide has the aminoacid sequence of SEQ ID NO:2. In another embodiment, the Ang-7polypeptide is a fragment, variant, derivative or analog of SEQ ID NO:2.The Ang-7 polypeptide, fragment, variant, derivative or analog isbiologically active. A biologically active Ang-7 polypeptide, fragment,variant, derivative or analog refers to the molecule's ability tomodulate angiogenesis such as, for example, by affecting endothelialtube formation, as described in, for example, the HUVEC assay in Example9 (infra), or by affecting tumor cell growth or proliferation (e.g., seeExample 10). Alternatively, such polypeptides, fragments, variants,derivatives or analogs which have the desired immunogenicity orantigenicity can be used in immunoassays, for immunization, forinhibition of Ang-7 polypeptide activity, and the like. Similarly, Ang-7fragments, variants, derivatives or analogs that retain, oralternatively lack or inhibit, a desired Ang-7 property of interest(e.g., inhibition of angiogenesis) can be used as inducers, orinhibitors of such property and its physiological correlates. A specificembodiment relates to an Ang-7 fragment that can be administered to asubject to inhibit angiogenesis. Fragments, variants, derivatives oranalogs of Ang-7 can be tested for the desired activity by proceduresknown in the art, including but not limited to the assays describedherein.

In another embodiment, an Ang-7 polypeptide, fragment, variant,derivative or analog has at least 10 contiguous amino acids. In otherembodiments, the Ang-7 polypeptide, fragment, variant, derivative oranalog consists of at least 20 or 50 contiguous amino acids. In anotherembodiment, the Ang-7 polypeptide, fragments, variants, derivatives oranalogs are not larger than 35, 100 or even 200 amino acids. Fragments,variants, derivatives and analogs of Ang-7 polypeptide include but arenot limited to those molecules comprising regions that are substantiallysimilar to an Ang-7 polypeptide (e.g., in various embodiments, at least60%, or 70%, or 80%, or 90%, or up to 95% identity over an amino acidsequence of identical size), or when compared to an aligned sequence inwhich the alignment is done by a computer sequence comparison/alignmentprogram known in the art, or when the encoding nucleic acid is capableof hybridizing to an ANG-7 nucleic acid, under stringent, moderatelystringent, or low stringency conditions.

Ang-7 polypeptide variants, derivatives or analogs can be made byaltering Ang-7 sequences by substitutions, additions or deletions thatprovide for functionally equivalent molecules. Ang-7 polypeptidevariants, derivatives or analogs include, but are not limited to, thosecontaining as a primary amino acid sequence of all or part of the aminoacid sequence of an Ang-7 polypeptide including altered sequences inwhich functionally equivalent amino acid residues (i.e., conservativesubstitutions) are substituted for residues within the sequence,resulting in a silent change. For example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofa similar polarity, hydrophobicity or hydrophilicity, which acts as afunctional equivalent, thereby resulting in a silent alteration.Substitutes for an amino acid within the sequence can be selected fromother members of the class to which the amino acid belongs.

Ang-7 polypeptide variants, fragments, derivatives and analogs can beproduced by various methods known in the art. The manipulations whichresult in their production can occur at the gene or protein level. Forexample, the cloned ANG-7 gene or cDNA sequence can be modified by anyof numerous strategies known in the art (see, e.g., Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The sequence can becleaved at appropriate sites with restriction endonuclease(s), followedby further enzymatic modification if desired, isolated, and ligated invitro. In the production of a nucleic acid encoding an Ang-7polypeptide, or fragment, variant, derivative or analog thereof, themodified nucleic acid remains in the proper translational reading frame,so that the reading frame is not interrupted by translational stopsignals or other signals which interfere with the synthesis. An ANG-7nucleic acid can also be mutated in vitro or in vivo to create and/ordestroy translation, initiation and/or termination sequences. Thenucleic acid sequence encoding an Ang-7 polypeptide can also be mutatedto create variations in coding regions and/or to form new restrictionendonuclease sites or destroy preexisting ones and to facilitate furtherin vitro modification. Any technique for mutagenesis known in the artcan be used, including but not limited to chemical mutagenesis, in vitrosite-directed mutagenesis (Hutchinson et al., J. Biol. Chem. 253:6551(1978)), the use of TAB® linkers (Pharmacia), and the like.

Manipulations of the Ang-7 polypeptide sequence can also be made at theprotein level. Included within the scope of the invention are Ang-7polypeptide variants, derivatives or analogs which are chemicallymodified during or after translation (e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, and the like). Any of numerouschemical modifications can be carried out by known techniques,including, but not limited to, specific chemical cleavage (e.g., bycyanogen bromide); enzymatic cleavage (e.g., by trypsin, chymotrypsin,papain, V8 protease, and the like); modification by, for example, NaBH₄,acetylation, formylation, oxidation and reduction, or metabolicsynthesis in the presence of tunicamycin, and the like.

In addition, Ang-7 polypeptides, or fragments, variants, derivatives andanalogs thereof can be chemically synthesized. For example, a peptidecorresponding to a portion, or fragment, of an Ang-7 polypeptide, whichcomprises a desired domain, or which mediates a desired activity invitro, can be synthesized by use of chemical synthetic methods using forexample an automated peptide synthesizer. Ang-7 polypeptide analogs canbe prepared, if desired, by introducing non-classical amino acids orchemical amino acid analogs as a substitution or addition into the Ang-7polypeptide sequence. Non-classical amino acids include, but are notlimited to, the D-isomers of the common amino acids, α-amino isobutyricacid, 4-aminobutyric acid, 2-amino butyric acid, γ-amino butyric acid,∈-Ahx, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionicacid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine,citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, selenocysteine, fluoro-amino acids,designer amino acids such as β-methyl amino acids, C α-methyl aminoacids, N α-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

In an embodiment, the Ang-7 polypeptide, or fragment, derivative oranalog thereof is a chimeric, or fusion protein, comprising an Ang-7polypeptide or variant, fragment, derivative or analog thereof(typically consisting of at least a domain or motif of the Ang-7polypeptide, or at least 10 contiguous amino acids of the Ang-7polypeptide) joined at its amino- or carboxy-terminus via a peptide bondto an amino acid sequence of a different protein. In one embodiment,such a chimeric protein is produced by recombinant expression of anucleic acid encoding the protein. The chimeric product can be made byligating the appropriate nucleic acid sequence, encoding the desiredamino acid sequences to each other by methods known in the art, in theproper coding frame and expressing the chimeric product by methodscommonly known in the art. Alternatively, the chimeric product can bemade by protein synthetic techniques (e.g., by use of an automatedpeptide synthesizer).

Expression of ANG-7 Nucleic Acids or Ang-7 Polypeptides

In a further embodiment, host cells comprise a construct expressing anANG-7 nucleic acid. Such a host cell can be a higher eukaryotic cell,such as a mammalian cell, a lower eukaryotic cell, such as a yeast cell,or a prokaryotic cell, such as a bacterial cell. Introduction of theconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE-dextran mediated transfection, or electroporation(Davis et al. Basic Methods in Molecular Biology, 2nd ed., Appleton andLang, Paramount Publishing, East Norwalk, Conn. (1994)), calciumchloride-mediated transformation, lithium acetate-mediatedtransformation, and the like.

The constructs in host cells can be used in a conventional manner toproduce the Ang-7 polypeptide, or fragment, variant, derivative oranalog thereof. Cell-free translation systems can also be employed toproduce such polypeptides using RNA's derived from the ANG-7 nucleicacid. Alternatively, the Ang-7 polypeptide, fragment, variant,derivative or analog thereof can be synthetically produced byconventional peptide synthesizers.

ANG-7 nucleic acids can be expressed in mammalian cells, yeast,bacteria, insect or other cells under the control of appropriatepromoters. Representative expression vectors include plasmid, phageand/or viral vector sequences, such as those described by Sambrook etal., Molecular Cloning: A Laboratory Manual (Second Edition, Cold SpringHarbor, N.Y., (1989)). For example, suitable vectors include adenoviralvectors, retroviral vectors, including lentiviral vectors, vacciniaviral vectors, cytomegalovirus viral vectors, and baculovirus vectors(see, e.g. Knops et a, J. Biol. Chem. 266:7285 (1991)), and the like.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early promoter, enhancer, splice, and polyadenylation sites, canbe used to provide the required nontranscribed genetic elements.Representative, useful vectors include pRc/CMV and pcDNA3 vectors(Invitrogen, San Diego, Calif.).

Promoters capable of directing the transcription of a nucleic acid canbe inducible or constitutive promoters and include viral and cellularpromoters. For expression in mammalian host cells, suitable viralpromoters include the immediate early cytomegalovirus promoter (Boshartet al., Cell 41:521-30 (1985)) and the SV40 promoter (Subramani et al.,Mol. Cell. Biol. 1:854-64 (1981)). Suitable cellular promoters forexpression of nucleic acids in mammalian host cells include, but are notlimited to the mouse metallothionien-1 promoter (Palmiter et al., U.S.Pat. No. 4,579,821), and tetracycline-responsive promoter (Gossen andBujard, Proc. Natl. Acad. Sci. USA 89:5547-51 (1992); Pescini et al.,Biochem. Biophys. Res. Comm. 202:1664-67 (1994)). Transcriptiontermination signals are also typically located downstream of the codingsequence of interest. Suitable transcription termination signals includethe early or late polyadenylation signals from SV40 (Kaufman and Sharp,Mol. Cell. Biol. 2:1304-19 (1982)), the polyadenylation signal from theAdenovirus 5 e1B region, and the human growth hormone gene terminator(DeNoto et al., Nucleic Acid. Res. 9:3719-30 (1981)).

Transcription of ANG-7 nucleic acids in mammalian cells is increased byinserting an enhancer sequence into the vector. Enhancers are cis-actingelements of DNA, usually about from 10 to 300 bp, that act on a promoterto increase its transcription. Examples include the SV40 enhancer on thelate side of the replication origin (bp 100 to 270), a cytomegalovirusearly promoter enhancer, a polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers.

Mammalian cells can be transfected by a number of methods includingcalcium phosphate precipitation (see, e.g., Wigler et al., Cell 14:725(1978); Corsaro and Pearson, Somatic Cell Genetics 7:603 (1981); Grahamand Van der Eb, Virology 52:456 (1973)); lipofection (see, e.g., Felgneret al., Proc. Natl. Acad. Sci. USA 84: 7413-17 (1987)) andmicroinjection and electroporation (see, e.g., Neumann et al., EMBO J.1: 841-45 (1982)). Mammalian cells can be transduced with virus such asSV40, CMV and the like. In the case of viral vectors, cloned DNAmolecules can be introduced by infection of susceptible cells with viralparticles. Retroviral, including lentiviral, and adenoviral vectors arepreferred for use in expressing ANG-7 nucleic acids in mammalian cells,particularly when ANG-7 nucleic acids or fragments, variants,derivatives or analogs thereof are used in methods of gene therapy.

Selectable markers are typically used to identify cells that contain theANG-7 nucleic acids. Selectable markers are generally introduced intothe cells along with the cloned DNA molecules and include genes thatconfer resistance to drugs, such as neomycin, hygromycin andmethotrexate. Selectable markers can also complement auxotrophies in thehost cell. Yet other selectable markers provide detectable signals, suchas β-galactosidase or green fluorescent protein, to identify cellscontaining ANG-7 nucleic acids. Selectable markers can be amplifiable.Such amplifiable selectable markers can be used to amplify the number ofsequences integrated into the host genome.

Various mammalian cell culture systems can be employed to express ANG-7nucleic acids. Examples of mammalian expression systems include theCOS-7 lines of monkey kidney fibroblasts (see, e.g., Gluzman, Cell23:175 (1981)), and other cell lines capable of expressing a compatiblevector, such as the C127, 3T3, CHO, HeLa, BHK, VERO, HeLa, MDCK, 293,W138, HEK, HUVEC cell lines. Once established, such cell lines can begrown in culture. Methods for culturing human cells in vitro and forimmortalizing cells are known to the skilled artisan.

For long-term, high-yield production of recombinant polypeptides, stableexpression is preferred. For example, cell lines which stably expressconstructs containing the ANG-7 nucleic acids can be engineered. Ratherthan using expression vectors that contain viral origins of replication,host cells can be transformed with ANG-7 nucleic acids, by appropriateexpression control elements (e.g., promoter and enhancer sequences,transcription terminators, polyadenylation sites, and the like), and aselectable marker. Following the introduction of such an expressionvector into mammalian cells, engineered cells can be allowed to grow for1-2 days in an enriched media, and then switched to a selective media.The selectable marker in the expression vector confers resistance to theselection and allows cells to stably integrate the vector into thechromosome and grow to form foci which in turn can be cloned andexpanded into cell lines.

A number of selection systems can be used, including, but not limited,to the herpes simplex virus thymidine kinase (“tk”) (see, e.g., Wigleret al., Cell 11:223 (1977)), hypoxanthine-guaninephosphoribosyltransferase (“hprt”) (sec, e.g., Szybalski et al., Proc.Natl. Acad. Sci. USA 48:2026 (1962)), and adeninephosphoribosyl-transferase genes (“aprt”) (see, e.g., Lowy et al., Cell22: 817 (1980)) and can be employed in tk⁻, hgprt⁻ or aprt⁻ cells,respectively. Antimetabolite resistance can also be used as the basis ofselection for dihydrofolate reductase (“dhfr”), which confers resistanceto methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA77:3567(1980); O'Hare et al., FEBS Lett. 210:731 (1981)); gpt, whichconfers resistance to mycophenolic acid (Mulligan et al., Proc. Natl.Acad. Sci. USA 78:2072. (1981)); neomcyin, which confers resistance tothe aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol. 150:1(1981)); and hygromycin, which confers resistance to hygromycin(Santerre et al., Gene 30:147 (1984)).

ANG-7 nucleic acids can also be expressed in Saccharomyces cerevisiae,Schizosaccharomyces prombe, filamentous fungi, and other single andmulticellular organisms that are amenable to transformation and/ortransfection. Methods for expressing cloned genes in Saccharomycescerevisiae are generally known in the art. (See, e.g., “Gene ExpressionTechnology” In Methods in Enzymology, Vol. 185, Goeddel (ed.), AcademicPress, San Diego, Calif. (1990); “Guide to Yeast Genetics and MolecularBiology” In Methods in Enzymology, Guthrie and Fink (eds.), AcademicPress, San Diego, Calif. (1991)). Filamentous fungi (e.g., strains ofAspergillus) can also be used to express the ANG-7 nucleic acids.Methods for expressing heterologous genes and cDNAs in culturedmammalian cells and in E. coli are discussed in detail in Sambrook etal. (Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989)). As would be evident to one skilled in the art, onecan express ANG-7 nucleic acids in other host cells such as avian,insect and plant cells using regulatory sequences, vectors and methodswell established in the literature.

Recombinant expression vectors useful for expression in bacterialtypically include origins of replication and selectable markerspermitting transformation of the host cell (e.g., the ampicillin ortetracycline resistance genes of E. coli or the TRP1 or URA3 gene of S.cerevisiae), and a promoter derived from a highly-expressed gene todirect transcription of a downstream structural sequence. Such promoterscan be derived from operons encoding glycolytic enzymes, such as3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, heatshock proteins, translation elongation factor, and the like. Theheterologous ANG-7 nucleic acid is assembled in appropriate phase withtranslation initiation and termination sequences, and preferably, aleader sequence capable of directing secretion of translated proteininto the periplasmic space or extracellular medium. Optionally, theheterologous sequence can encode a fusion protein including anN-terminal identification peptide imparting desired characteristics(e.g., stabilization or simplified purification of expressed recombinantproduct). Suitable prokaryotic hosts for transformation includeEscherichia coli, Bacillus subtilis, Salmonella typhimurium and variousspecies within the genera Pseudomonas, Streptomyces, and Staphylococcus,although others can also be employed as a routine matter of choice.

Useful expression vectors for bacterial use comprise a selectable markerand bacterial origin of replication derived from plasmids comprisinggenetic elements of the well-known cloning vector pBR322 (ATCC 37017).Other vectors include but are not limited to, pBLUESCRIPT vectors(Stratagene), PKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) andGEM1 (Promega Biotec, Madison, Wis.). These pBR322 “backbone” sectionsare combined with an appropriate promoter and the structural sequence tobe expressed.

Useful expression vectors further comprise a fusion partner for ease inpurifying a desired polypeptide or for producing soluble polypeptides.Examples of commercial fusion vectors include but are not limited topET32a (Novagen, Madison, Wis.), pGEX-4T-2 (Pharmacia) and pCYB3 (NewEngland Biolabs, Beverly, Mass.). Expression vectors which avoid the useof fusion partners can also be constructed particularly for high levelexpression of Ang-7 polypeptides, or fragments, variants, derivatives oranalogs thereof in bacterial cells. For example, vectors can be made tooptimize translational coupling, as described by Pilot-Matias et al.(Gene 128:219-225 (1993)). Alternatively, an ANG-7 nucleic acid can beco-expressed with a separate accessory plasmid which itself encodes aprotein or peptide that aids in solubilizing an Ang-7 polypeptide ofinterest. (See, e.g., Makrides, Microbiological Reviews 60:512 (1996)).

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction), and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification. Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents; suchmethods are well-known to the ordinary artisan.

Ang-7 polypeptides can be isolated using a number of establishedmethods, such as affinity chromatography using anti-Ang-7 antibodiescoupled to a solid support and sequence-specific chromatography asdescribed by Lobanenkov et al. (Oncogene 5:1743-53 (1990)) and usingantibodies against an epitope-tagged Ang-7 polypeptide (e.g., anti-HIS₄,myc, FLAG, and the like). Additional isolation methods includepurification means such as liquid chromatography, high pressure liquidchromatography, FPLC, gradient centrifugation and gel electrophoresis,among others. Methods of protein purification are known in the art andcan be applied to the purification of recombinant polypeptides describedherein. (See generally, Scopes, Protein Purification, Springer-Verlag,NY (1982)).

Anti-Ang-7 Antibodies

In another embodiment, the invention provides anti-Ang-7 antibodies foruse in modulating angiogenesis. Such antibodies can bind to Ang-7polypeptides, or fragments, variants, derivatives or analogs thereof.Ang-7 polypeptides can be used to raise antisera or monoclonalantibodies following, for example, the method of Kohler and Milstein(Nature 256:495 (1975)). Such monoclonal antibodies can then form thebasis for a treatment, therapeutic use, or diagnostic test.

The production of non-human antisera or monoclonal antibodies (L&,murine, lagormorpha, porcine or equine) can be accomplished by, forexample, immunizing an animal with Ang-7 polypeptides, fragments,variants, derivatives or analogs, with or without an adjuvant. For theproduction of monoclonal antibodies, antibody producing cells areobtained from immunized animals, immortalized and screened, or screenedfirst for the production of the antibody that binds to the antigen, andthen immortalized. It can be desirable to transfer the antigen bindingregions (e.g., F(ab′), F(ab′)₂, Fv, or hypervariable regions) ofnon-human antibodies into the framework of a human antibody byrecombinant DNA techniques to produce a substantially human molecule.Methods for producing such “humanized” molecules are generally wellknown and described in, for example, U.S. Pat. Nos. 4,816,567;4,816,397; 5,693,762; and 5,712,120; International Patent Publication WO87/02671 and WO 90/00616; and European Patent Publication 0,239,400; thedisclosures of which are incorporated by reference herein).Alternatively, a human monoclonal antibody or portions thereof can beidentified by first screening a human B-cell cDNA library for DNAmolecules that encode antibodies that specifically bind to an Ang-7polypeptide according to the method generally set forth by Huse et al.(Science 246:1275-81 (1989)). The DNA molecule can then be cloned andamplified to obtain sequences that encode the antibody (or bindingdomain) of the desired specificity. Phage display technology offersanother technique for selecting antibodies that bind to Ang-7polypeptides. (See, e.g., International Patent Publications WO 91/17271and WO 92/01047, and Huse et al., supra).

Antibodies can also be produced by genetic immunization using expressionvectors to direct the expression of Ang-7 polypeptides. Particlebombardment-mediated gene transfer (Tang et al., Nature 356:152-54(1992); Eisenbaum et al., DNA & Cell Biol. 12:791-97 (1993); Johnstonand Tang, Meth. Cell Biol. 43 Pt.A:353-65 (1994); Vahlsing et al., J.Immun. Meth. 175:11-22 (1994)) and retroviral gene transfer (Wang et a,DNA & Cell Biol. 12:799-805 (1993); Stover, Curr. Opin. Immunol.6:568-71 (1994); Laube et al., Human Gene Ther. 5:853-62 (1994)) havebeen used to generate specific antibody responses to proteins encoded bytransferred genes. These methods permit the production of antibodieswithout requiring protein purification. Such methods can be used toproduce panels of antibodies specific to Ang-7 polypeptides. Monoclonalantibodies can also be generated using these methods.

Antibodies against Ang-7 polypeptides can be used as reagents to detectAng-7 polypeptides in biological samples, such as tumor biopsy samples,tissue and organ sections, peripheral blood cells, and the like. Suchantibodies can also be used in immunoassays to detect and/or quantitateAng-7 polypeptide levels. Immunoassays suitable for use in the presentinvention include, but are not limited to, enzyme-linked immunosorbantassays, immunoblots, inhibition or competition reactions, sandwichassays, radioimmunoprecipitation, and the like. (See, e.g., U.S. Pat.Nos. 4,642,285; 4,376,110; 4,016,043; 3,879,262; 3,852,157; 3,850,752;3,839,153; 3,791,932; and Harlow and Lane, Antibodies, A LaboratoryManual, Cold Spring Harbor Publications, NY (1988)).

In one assay, Ang-7 polypeptides are identified and/or quantified usinglabeled antibodies, preferably labeled monoclonal antibodies. Theantibodies are reacted with tissues or cells, and then the tissues orcells are examined to determine whether the antibodies specificallybound to the target Ang-7 polypeptide. Such assays are typicallyperformed under conditions conducive to immune complex formation.Unlabeled primary antibody can be used in combination with labels thatare reactive with primary antibody to detect the Ang-7 polypeptide. Forexample, the primary antibody can be detected indirectly by a labeledsecondary antibody made to specifically detect the primary antibody.Alternatively, the anti-Ang-7 antibody can be directly labeled. A widevariety of labels can be employed, such as radionuclides, particles (I,gold, ferritin, magnetic particles and red blood cells), fluorophores,chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzymeinhibitors, ligands (particularly haptens), and the like.

The anti-Ang-7 polypeptides can be used in a diagnostic assay fordetecting levels of polypeptides of the present invention, for example,in various tissues, since an under-expression of the proteins comparedto normal control tissue samples may detect the presence of abnormalangiogenesis, for example, a tumor. Assays used to detect levels ofprotein in a sample derived from a host are well-known to those of skillin the art and include radioimmunoassays, competitive-binding assays,Western blot analyses, ELISA assays and “sandwich” type assays.Diagnostic assays also include the detection of polynucleotides whichcode for the polypeptides of the present invention.

Methods of Using ANG-7 Nucleic Acids and/or Ang-7 Polypeptides:

In another embodiment, methods and compositions are provided for theadministration of an Ang-7 compound to modulate angiogenesis. Ang-7compounds include, but are not limited to, Ang-7 polypeptides,fragments, variants, derivatives and analogs thereof, as describedherein. Such Ang-7 compounds can further include ANG-7 nucleic acidsencoding Ang-7 polypeptide, fragments or variants, as described herein,and ANG-7 antisense nucleic acids, as more fully described below.Disorders involving angiogenesis, such as unwanted angiogenesis, use anAng-7 compound that inhibits angiogenesis, such as the administration ofAng-7 polypeptides, fragments, variants, derivatives and/or analogsthereof and/or ANG-7 nucleic acids. Similarly, disorders in whichangiogenesis is deficient or is desired can be treated by administrationan ANG-7 antisense nucleic acid, or an Ang-7 polypeptide, a fragment,variant, derivative or analog thereof that inhibits Ang-7 function. Inanother embodiment, Ang-7 compounds include antibodies, such aspolyclonal, monoclonal and humanized antibodies.

The compounds can be administered therapeutically or prophylactically.They can be contacted with the host cell in vivo, ex vivo, or in vitro,in an effective amount, as demonstrated by the following examples.

Gene Therapy

ANG-7 nucleic acids coding for Ang-7 polypeptides of the presentinvention, can be used in a process of gene therapy. Gene therapy refersto the process of providing for the expression of nucleic acid sequencesof exogenous origin in a subject for the treatment of a disease orclinical condition within that subject. Such gene therapy can beinvolved in the treatment of a disease or clinical condition which caninclude, but is not limited to, cancer, wound healing, diabeticretinopathies, macular degeneration, cardiovascular diseases, andclinical conditions involving angiogenesis in the reproductive system,including regulation of placental vascularization or use as anabortifacient. Delivery of the nucleic acid into a subject can be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid-carrying vector, or indirect, in which case cellsare first transformed with the nucleic acid in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo, or ex vivo gene therapy. For example, Ang-7 polypeptide, or afragment or variant thereof, can be recombinantly expressed byengineering cells with a polynucleotide (DNA or RNA) coding for thepolypeptide, fragment or variant ex vivo, the engineered cells are thenprovided to a patient to be treated with the polypeptide. Cells can alsobe engineered by procedures known in the art by use of aretroviral/lentiviral particle containing RNA encoding the Ang-7polypeptide, fragment of variant. Exemplary methods are described below.

For general reviews of the methods of gene therapy, see Goldspiel et al.(Clinical Pharmacy 12:488-505 (1993)); Wu and Wu (Biotherapy 3:87-95(1991)); Tolstoshev (Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993));Mulligan (Science 260:926-932 (1993)); Morgan and Anderson (Ann. Rev.Biochem. 62:191-217 (1993)); and May (TIBTECH 11:155-215 (1993)).Methods commonly known in the art of recombinant DNA technology that canbe used include those described in Ausubel et al. (Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993)); Kriegler (GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY(1990)); and U.S. Pat. Nos. 6,077,663; 6,077,835; 6,077,705; and6,075,012. Methods using other exogenous sequences for gene therapy arealso applicable to gene therapy using ANG-7 nucleic acids. (See A, U.S.Pat. No. 6,066,624).

Several methods for transferring potentially therapeutic genes todefined cell populations are known. (e, e.g. Mulligan, Science260:926-31 ((1993).) These methods include:

1) Direct gene transfer. (See, e.g., Wolff et al., Science 247:1465-68(1990)).

2) Liposome-mediated DNA transfer. (See, e.g., Caplen et al., NatureMed, 3:39-46 (1995); Crystal, Nature Med. 1:15-17 (1995); Gao and Huang,Biochem. Biophys. Res. Comm. 179:280-85 (1991)).

3) Retrovirus-mediated DNA transfer. (See, e.g., Kay et al., Science,262:117-19 (1993); Anderson, Science 256:808-13 (1992)). Retrovirusesfrom which the retroviral plasmid vectors hereinabove mentioned can bederived include lentiviruses. They further include, but are not limitedto, Moloney Murine Leukemia Virus, spleen necrosis virus, retrovirusessuch as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus. Examples illustrating the use of retroviral vectors ingene therapy further include the following: Clowes et al. (J. Clin.Invest. 93:644-651 (1994)); Kiem et al. (Blood 83:1467-1473 (1994));Salmons and Gunzberg (Human Gene Therapy 4:129-141 (1993)); and Grossmanand Wilson (Curr. Opin. in Genetics and Devel. 3:110-114 (1993)).

4) DNA Virus-mediated DNA transfer. Such DNA viruses includeadenoviruses (preferably Ad-2 or Ad-5 based vectors), herpes viruses(preferably herpes simplex virus based vectors), and parvoviruses(preferably “defective” or non-autonomous parvovirus based vectors, morepreferably adeno-associated virus based vectors, most preferably AAV-2based vectors). (See, e.g. Ali et al., Gene Therapy 1:367-84 (1994);U.S. Pat. Nos. 4,797,368 and 5,139,941, the disclosures of which areincorporated herein by reference.) Adenoviruses have the advantage thatthey have a broad host range, can infect quiescent or terminallydifferentiated cells, such as neurons or hepatocytes, and appearessentially non-oncogenic. Adenoviruses do not appear to integrate intothe host genome. Because they exist extrachromosomally, the risk ofinsertional mutagenesis is greatly reduced. Adeno-associated virusesexhibit similar advantages as adenoviral-based vectors. However, AAVsexhibit site-specific integration on human chromosome 19.

Kozarsky and Wilson (Current Opinion in Genetics and Development3:499-503 (1993)) present a review of adenovirus-based gene therapy.Bout et al. (Human Gene Therapy 5:3-10 (1994)) demonstrated the use ofadenovirus vectors to transfer genes to the respiratory epithelia ofrhesus monkeys. Herman et al. (Human Gene Therapy 10:1239-1249 (1999))describe the intraprostatic injection of a replication-deficientadenovirus containing the herpes simplex thymidine kinase gene intohuman prostate, followed by intravenous administration of the prodrugganciclovir in a phase I clinical trial. Other instances of the use ofadenoviruses in gene therapy can be found in Rosenfeld et al. (Science252:431-434 (1991)); Rosenfeld et al. (Cell 68:143-155 (1992));Mastrangeli et al. (J. Clin. Invest. 91:225-234 (1993)); and Thompson(Oncol. Res. 11:1-8 (1999)).

The choice of a particular vector system for transferring the gene ofinterest will depend on a variety of factors. One important factor isthe nature of the target cell population. Although retroviral vectorshave been extensively studied and used in a number of gene therapyapplications, these vectors are generally unsuited for infectingnon-dividing cells. In addition, retroviruses have the potential foroncogenicity. However, recent developments in the field of lentiviralvectors may circumvent some of these limitations. (See Naldini et al.,Science 272:263-7 (1996).)

Gene therapy with DNA encoding a polypeptide of the present invention isprovided to a subject (e.g., a patient or mammal) in need thereof,concurrent with, or immediately after diagnosis. The skilled artisanwill appreciate that any suitable gene therapy vector containing DNAencoding a polypeptide of the present invention can be used inaccordance with the present invention. The techniques for constructingsuch a vector are known. (See, e.g., Anderson, Nature 392 25-30 (1998);Verma, Nature 389 239-42 (1998)). Introduction of the vector to thetarget site can be accomplished using known techniques.

In one embodiment, ANG-7 nucleic acid is inserted into an expressionvector. The ANG-7 nucleic acids encode an Ang-7 polypeptide, fragment,variant, derivative or chimeric protein. In particular, such anexpression vector construct typically comprises a promoter operablylinked to an ANG-7 nucleic acid (e.g. cDNA or a portion of the codingregion), the promoter being inducible or constitutive, and, optionally,tissue-specific.

In another embodiment, if an endogenous ANG-7 nucleic acid is defective,the defective sequences can be replaced by exogenous ANG-7 codingsequences and any other desired sequences that are flanked by regionsthat promote homologous recombination at a desired site in the genome,thus providing for intrachromosomal expression of the exogenous ANG-7nucleic acid. (See e.g., Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)); U.S.Pat. Nos. 5,631,153; 5,627,059; 5,487,992; and 5,464,764.).

Nucleic acids can also administering in linkage to a peptide which isknown to enter the nucleus, by administering the nucleic acid in linkageto a ligand subject to receptor-mediated endocytosis (see e.g., Wu andWu, J. Biol. Chem. 262:4429-4432 (1987)), which can be used to targetcell types specifically expressing the receptors, and the like. Inanother embodiment, a nucleic acid-ligand complex can be formed in whichthe ligand comprises a fusogenic viral peptide to disrupt endosomes,allowing the nucleic acid to avoid lysosomal degradation.

In yet another embodiment, the nucleic acid can be targeted in vivo forcell specific uptake and expression, by targeting a specific receptor(e.g., International Patent Publications WO 92/06180; WO 92/22635; WO92/20316; WO 93/14188, and WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression by homologous recombination. (See, e.g., Koller andSmithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra etal., Nature 342:435438 (1989); U.S. Pat. Nos. 5,631,153; 5,627,059;5,487,992; and 5,464,764).)

In a specific embodiment, a viral vector is used that contains an ANG-7nucleic acid. For example, a retroviral vector can be used (see Milleret al., Meth. Enzymol. 17:581-599 (1993)). These retroviral vectors havebeen modified to delete retroviral sequences that are not necessary forpackaging of the viral genome and integration into host cell DNA. TheANG-7 nucleic acid to be used in gene therapy is cloned into the vector,which facilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al. (Biotherapy 6:291-302(1994)), which describes the use of a retroviral vector to deliver themolrl gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy.

Other approaches to gene therapy involve transferring a gene to cells intissue culture by methods such as electroporation, lipofection, calciumphosphate mediated transfection, or viral infection. Typically, themethod includes the transfer of a selectable marker to the cells. Thecells are then placed under selection to isolate those cells that havetaken up and are expressing the transferred gene. The selected cells arethen delivered to a patient.

Numerous techniques are known in the art for the introduction of foreigngenes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92 (1985)) and can be used in accordancewith the present invention. The technique typically provides for thestable transfer of the nucleic acid to the cell, so that the nucleicacid is expressible by the cell and is heritable and expressible by itscell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Typically, cells are injected subcutaneously.Alternatively, recombinant skin cells can be applied as a skin graftonto the patient. Recombinant blood cells (e.g., hematopoietic stem orprogenitor cells) are typically administered intravenously. The amountof cells envisioned for use depends on the desired effect, the patient'scondition, and the like, and can be determined by one skilled in theart.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to endothelial cells, prostate cells, epithelial cells,keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells (suchas T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes and granulocytes), various stem orprogenitor cells (in particular, hematopoietic stem or progenitor cells,such as those obtained from bone marrow, umbilical cord blood,peripheral blood, fetal liver, and the like). The cells used for genetherapy generally are autologous to the patient, but heterologous cellsthat can be typed for compatibility with the patient can be used.

Administration of Ang-7 Polypeptides, or Fragments, Variants,Derivatives or Analogs Thereof:

The invention provides methods for the administration to a subject of aneffective amount of an Ang-7 compound. For example, unwantedangiogenesis can be treated or prevented by administration of aneffective amount of Ang-7 polypeptide, fragment, variant, derivative oranalog thereof. In one embodiment, such polypeptides are administeredtherapeutically (including prophylactically) in diseases or clinicalcondition involving increased (relative to normal or desired)angiogenesis, to thereby inhibit angiogenesis. In another embodiment,such polypeptides are administered therapeutically (includingprophylactically) in diseases or clinical conditions where angiogenesismay be relevant to the causation or treatment of the disease or clinicalcondition in order to inhibit the disease or clinical condition. Thediseases or clinical conditions of the present invention include but arenot limited to, cancer, wound healing, tumor formation, diabeticretinopathies, macular degeneration, cardiovascular diseases, and thelike.

Typically, the Ang-7 compound is substantially purified prior toformulation. The subject can be an animal, including but not limited to,cows, pigs, horses, chickens, cats, dogs, and the like, and is typicallya mammal, and in a particular embodiment human. In another specificembodiment, a non-human mammal is the subject. Formulations and methodsof administration that can be employed when the Ang-7 compound comprisesa nucleic acid are described above; additional appropriate formulationsand routes of administration can be selected from among those describedbelow.

Various delivery systems are known and can be used to administer anAng-7 compound, such as, for example, encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of an expression vectorcomprising an ANG-7 nucleic acid as part of a retroviral or othervector, and the like. Methods of introduction include but are notlimited to, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural and oral routes. The compounds can beadministered by any convenient route, for example, by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa) and the like, and canbe administered together with other biologically active agents.Administration can be systemic or local.

In a specific embodiment, it can be desirable to administer an Ang-7compound locally to the area in need of treatment; this administrationcan be achieved by, for example, and not by way of limitation, localinfusion during surgery, topical application (e.g., in conjunction witha wound dressing after surgery), by injection, by means of a catheter,by means of a suppository, or by means of an implant, the implant beingof a porous, non-porous, or gelatinous material, including membranessuch as sialastic membranes, or fibers. In one embodiment,administration can be by direct injection at the site (or former site)of a malignant tumor or neoplastic or pre-neoplastic tissue.

In another embodiment, the Ang-7 compound can be delivered in a vesicle,in particular a liposome (see Langer, Science 249:1527-1533 (1990);Treat et al., In Liposomes in the Therapy of Infectious Disease andCancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365(1989); Lopez-Berestein, supra, pp. 317-327); U.S. Pat. Nos. 6,077,663and 6,071,533).

In yet another embodiment, the Ang-7 compound can be delivered in acontrolled release system. In one embodiment, a pump can be used (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al, Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(et, al., Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228:190 (1985);During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg.71:105 (1989)). In yet another embodiment, a controlled release systemcan be placed in proximity of the therapeutic target, thus requiringonly a fraction of the systemic dose (see e.g., Goodson, in MedicalApplications of Controlled Release, supra, Vol. 2, pp. 115-138 (1984)).Other controlled release systems are discussed in, for example, thereview by Langer (Science 249:1527-1533 (1990)).

The present invention also provides pharmaceutical compositions foradministering Ang-7 compounds. Such compositions comprise atherapeutically effective amount of an Ang-7 compound and apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more typically inhumans. The term “carrier” refers to a diluent, adjuvant, excipient,stabilizer, or vehicle with which the Ang-7 compound is formulated foradministration.

A pharmaceutically acceptable carrier can contain a physiologicallyacceptable compound that acts, for example, to stabilize the compositionor to increase or decrease the absorption of the agent. Aphysiologically acceptable compound can include, for example,carbohydrates, such as glucose, sucrose or dextrans, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins or other stabilizers or excipients. Other physiologicallyacceptable compounds include wetting agents, emulsifying agents,dispersing agents or preservatives, which are particularly useful forpreventing the growth or action of microorganisms. Carriers furtherinclude sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil, and the like. Water is a typicalcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Various preservatives are well known and include,for example, phenol and ascorbic acid. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol, and the like. One skilled in the art would know that thechoice of a pharmaceutically acceptable carrier, including aphysiologically acceptable compound, depends, for example, on the routeof administration of the polypeptide and on the particularphysio-chemical characteristics of the specific polypeptide. Forexample, a physiologically acceptable carrier such as aluminummonosterate or gelatin is particularly useful as a delaying agent, whichprolongs the rate of absorption of a pharmaceutical compositionadministered to a subject. Further examples of carriers, stabilizers oradjutants can be found in Martin, Remington's Pharm. Sci., 15th Ed.(Mack Publ. Co., Easton, 1975), incorporated herein by reference. Thepharmaceutical composition also can be incorporated, if desired, intoliposomes, microspheres or other polymer matrices (See Gregoriadis,Liposome Technology, Vol. 1 (CRC Press, Boca Raton, Fla. 1984), which isincorporated herein by reference). Liposomes, for example, which consistof phospholipids or other lipids, are nontoxic, physiologicallyacceptable and metabolizable carriers that are relatively simple to makeand administer.

When practiced in vivo, methods of administering a pharmaceuticalcomposition containing the vector of this invention, are well known inthe art and include but are not limited to, administration orally,intra-tumorally, intravenously, intramuscularly or intraperitoneal.Administration can be effected continuously or intermittently and willvary with the subject and the condition to be treated, for example, asis the case with other therapeutic compositions (see Landmann et al., J.Interferon Res. 12:103-111 (1992); Aulitzky et al, Eur. J. Cancer27:462-67 (1991); Lantz et al., Cytokine 2:402-06(1990); Supersaxo etal., Pharm. Res. 5:472-76(1988); Demetri et al., J. Clin. Oncol.7:1545-53 (1989); and LeMaistre et al., Lancet 337:1124-25 (1991)).

Pharmaceutical compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations, and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulations can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, and the like.

Pharmaceutical compositions will contain a therapeutically effectiveamount of the Ang-7 compound, typically in purified form, together witha suitable amount of carrier so as to provide a formulation proper foradministration to the patient. The formulation should suit the mode ofadministration.

In one embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition can also include a solubilizingagent and a local anesthetic to ease pain at the site of the injection.Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form. For example, as a dry lyophilized powderor water-free concentrate in a hermetically sealed container such as anampoule or sachette indicating the quantity of Ang-7 compound. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water orsaline. Where the composition is administered by injection, an ampouleof sterile water for injection or saline can be provided so that theingredients can be mixed prior to administration.

The Ang-7 compounds can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, and the like, and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The amount of the Ang-7 compound that will be effective in the treatmentof a particular disorder or condition as indicated by modulation ofangiogenesis will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. In addition, in vitroassays can optionally be employed to help identify optimal dosageranges. The precise dose of the Ang-7 compound to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Suitable dosage ranges for intravenous administration are generallyabout 20-500 micrograms of active Ang-7 compound per kilogram bodyweight. Suitable dosage ranges for intranasal administration aregenerally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effectivedoses can be extrapolated from dose response curves derived from invitro or animal model test systems. Suppositories generally containactive ingredient in the range of 0.5% to 10% by weight; oralformulations typically contain 10% to 95% active ingredient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Treatment of Angiogenesis-Related Diseases

In another embodiment, Ang-7 compounds, such as Ang-7 polypeptides, orfragments, variants, derivatives or analogs thereof, or ANG-7 nucleicacids encoding such polypeptides, can be used to treat diseases orclinical conditions that may be related to angiogenesis. Withoutintending to be bound by any particular theory, it is believed that theinitiation and/or progression of many diseases is dependent onangiogenesis. For example, tumor formation is closely associated withangiogenesis. Such tumors include solid tumors, such asrhabdomyosarcomas, retinoblastoma, Ewing sarcoma, neuroblastoma, andosteosarcoma, and benign tumors, such as acoustic neuroma, neurofibroma,trachoma and pyogenic granulomas. Thus, tumor formation or progressioncan be treated by inhibiting angiogenesis; administering Ang-7polypeptides, fragments, variants, derivatives and analogs, or ANG-7nucleic acids, to the tumor will inhibit further tumor growth and/orprogression. Similarly, cells expressing recombinant Ang-7 polypeptides,fragments or variants can be used. Alternatively, decreased angiogenesisis associated with cardiovascular disease. Thus, Ang-7 compounds thatincrease angiogenesis can be used to treat angiogenesis. Any of themethodologies described above can be applied to the treatment of suchangiogenesis-related diseases.

Other diseases or clinical conditions involving unwanted angiogenesiscan also be treated in a similar manner. Such other diseases orconditions include, but are not limited to, ocular neovascular disease,age-related macular degeneration, diabetic retinopathy, corneal graftrejection, neovascular glaucoma and retrolental fibroplasia, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium, keratitis sicca,Sjogren's syndrome, acne rosacea, phylectenulosis, syphilis,Mycobacteria infections, lipid degeneration, chemical burns, bacterialulcers, fungal ulcers, Herpes simplex infections, Herpes zosterinfections, protozoan infections, Kaposi sarcoma, Mooren ulcer,Terrien's marginal degeneration, mariginal keratolysis, rheumatoidarthritis, systemic lupus, polyarthritis, Wegener's sarcoidosis,Scleritis, Steven's Johnson disease, periphigoid radial keratotomy,corneal graph rejection, sickle cell anemia, sarcoidosis, syphilis,pseudoxanthoma elasticum, Pagets disease, vein occlusion, arteryocclusion, carotid obstructive disease, chronic uveitis/vitreitis,Lyme's disease, systemic lupus erythematosis, retinopathy ofprematurity, Eales disease, Bechets disease, infections causing aretinitis or choroiditis, presumed ocular histoplasmosis, Bests disease,myopia, optic pits, Stargart's disease, pars planitis, chronic retinaldetachment, hyperviscosity syndromes, toxoplasmosis, trauma andpost-laser complications. Other diseases include those associated withrubeosis, abnormal proliferation of fibrovascular or fibrous tissue,rheumatoid arthritis, osteoarthritis, ulcerative colitis, Crohn'sdisease, Bartonellosis, atherosclerosis, hemangioma, Osler-Weber-Rendudisease, hereditary hemorrhagic telangiectasia, and leukemia.

Antisense Regulation of ANG-7 Expression:

In a specific embodiment, Ang-7 function is inhibited by use of ANG-7antisense nucleic acids. The present invention provides for theadministration of nucleic acids of at least six nucleotides that areantisense to a gene or cDNA encoding Ang-7 polypeptide or a fragment orvariant thereof to inhibit the function of Ang-7 polypeptide. An ANG-7“antisense” nucleic acid as used herein refers to a nucleic acid thathybridizes to a portion of an ANG-7 RNA (typically mRNA) by virtue ofsome sequence complementarity. The antisense nucleic acid can becomplementary to a coding and/or noncoding region of an ANG-7 mRNA.Absolute complementarity, although typical, is not required, however. Asequence “complementary to at least a portion of an RNA,” as referred toherein, means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex. In the case ofdouble-stranded ANG-7 antisense nucleic acids, a single strand of theduplex DNA can be tested, or triplex formation can be assayed. Theability to hybridize will depend on both the degree of complementarityand the length of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches it can contain andstill form a stable duplex (or triplex, as the case may be). One skilledin the art can ascertain a tolerable degree of mismatch by use ofstandard procedures to determine the melting point of the hybridizedcomplex.

Such antisense nucleic acids have utility as agents that inhibit Ang-7function, and can be used in the treatment or prevention of diseases orclinical conditions, as described supra. The antisense nucleic acids ofthe invention can be oligonucleotides that are double-stranded orsingle-stranded, RNA or DNA, or a derivative thereof, which can bedirectly administered to a cell, or which can be producedintracellularly by transcription of exogenous, introduced nucleic acidsequences.

In a specific embodiment, the ANG-7 antisense nucleic acid provided bythe instant invention can be used to prevent angiogenesis. The inventionfurther provides pharmaceutical compositions comprising an effectiveamount of the ANG-7 antisense nucleic acids of the invention in apharmaceutically acceptable carrier, as described supra. In anotherembodiment, the invention is directed to methods for inhibiting theexpression of an ANG-7 nucleic acid sequence in a eukaryotic cellcomprising providing the cell with an effective amount of a compositioncomprising an ANG-7 antisense nucleic acid of the invention. ANG-7antisense nucleic acids and their uses are described in detail below.

The ANG-7 antisense nucleic acids are of at least six nucleotides andare typically oligonucleotides (ranging from 6 to about 50 nucleotidesor more). In specific aspects, the oligonucleotide is at least 10nucleotides, at least 15 nucleotides, at least 100 nucleotides, or canbe at least 200 nucleotides. The oligonucleotides can be DNA or RNA orchimeric mixtures or derivatives thereof, and can be single-stranded ordouble-stranded. A derivative can be modified at the base moiety, sugarmoiety, or phosphate backbone, as described below. The derivative caninclude other appending groups such as peptides, or agents facilitatingtransport across the cell membrane (see e.g., Letsinger et al., Proc.Natl. Acad. Sci. USA 86:6553-56 (1989); Lemaitre et al., Proc. Natl.Acad. Sci. USA 84:648-52 (1987); International Patent Publication WO88/09810) or blood-brain barrier (see, e.g., International PatentPublication WO 89/10134), hybridization-triggered cleavage agents (see,e.g., Krol et al., BioTechniques 6:958-76 (1988)) or intercalatingagents (see, e.g., Zon, Pharm. Res. 5:539-49 (1988)).

In one embodiment of the invention, a ANG-7 antisense oligonucleotide isprovided, typically as single-stranded DNA. The oligonucleotide can bemodified at any position on its structure with substituents generallyknown in the art. The ANG-7 antisense oligonucleotide can comprise atleast one modified base moiety, such as, for example, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxy-hydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylamino-methyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, 2,6-diaminopurine, and the like.In another embodiment, the oligonucleotide comprises at least onemodified sugar moiety, such as, for example, arabinose,2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the oligonucleotide comprises at least onemodified phosphate backbone, such as, for example, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

In yet another embodiment, the oligonucleotide is an α-anomericoligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (see Gautier etal., Nucl. Acids Res. 15:6625-41 (1987)). The oligonucleotide can beconjugated to another molecule (e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, hybridization-triggered cleavageagent, and the like).

Oligonucleotides of the invention can be synthesized by standard methodsknown in the art (e.g., by use of a commercially available automated DNAsynthesizer). As examples, phosphorothioate oligonucleotides can besynthesized by the method of Stein et al. (see Nucl. Acids Res. 16:3209(1988)), and methyphosphonate oligonucleotides can be prepared by use ofcontrolled pore glass polymer supports (see Sarin et al., Proc. Natl.Acad. Sci. USA 85:7448-51 (1988)), and the like.

In a specific embodiment, the ANG-7 antisense oligonucleotide comprisescatalytic RNA, or a ribozyme (see, e.g., International PatentPublication WO 90/11364; Sarver et al., Science 247:1222-25 (1990)). Inanother embodiment, the oligonucleotide is a 2′-0-methylribonucleotide(see e.g., Inoue et al., Nucl. Acids Res. 15:6131-48 (1987)), or achimeric RNA-DNA analogue (see e.g., Inoue et al, FEBS Lett. 215:327-30(1987)).

In an alternative embodiment, the ANG-7 antisense nucleic acid of theinvention is produced intracellularly by transcription from an exogenoussequence. For example, a vector can be introduced in vivo such that itis taken up by a cell, within which the vector or a portion thereof istranscribed, producing an antisense nucleic acid (RNA) of the invention.The vector would contain a sequence encoding the ANG-7 antisense nucleicacid or a portion thereof. Once inside the cell the vector can remainepisomal or become chromosomally integrated, as long as it can betranscribed to produce the desired antisense RNA. Such vectors can beconstructed by recombinant DNA technology methods standard in the art.Vectors can be plasmid, viral, or others known in the art and used forreplication and expression in mammalian cells. Expression of thesequence encoding the ANG-7 antisense RNA can be controlled by anypromoter known in the art to act in mammalian, typically human, cells.The promoters can be inducible or constitutive. Inducible promotersinclude but are not limited to, the SV40 early promoter region (seeBernoist and Chambon, Nature 290:304-10 (1981)), the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (see Yamamoto etal., Cell 22:787-97 (1980)), the herpes thymidine kinase promoter (seeWagner et al., Proc. Natl. Acad. Sci. USA 78:1441-45(1981)), theregulatory sequences of the metallothionein gene (see Brinster et al.,Nature 296:39-42(1982)), and the like.

Animal Models:

The invention also provides animal models. In one embodiment, animalmodels for diseases and disorders involving angiogenesis are provided.Such an animal can be initially produced by promoting homologousrecombination between an ANG-7 gene in its chromosome and an exogenousANG-7 gene that has been rendered biologically inactive (typically byinsertion of a heterologous sequence, such as an antibiotic resistancegene). For example, homologous recombination is carried out bytransforming embryo-derived stem (ES) cells with a vector containing theinsertionally inactivated ANG-7 gene, such that homologous recombinationoccurs, followed by injecting the ES cells into a blastocyst, andimplanting the blastocyst into a foster mother, followed by the birth ofthe chimeric animal (“knockout animal”) in which an ANG-7 gene has beeninactivated (see, e.g., Capecchi, Science 244:1288-1292 (1989); U.S.Pat. Nos. 5,631,153; 5,627,059; 5,487,992; and 5,464,764). The chimericanimal can be bred to produce additional knockout animals. Such animalscan be mice, hamsters, sheep, pigs, cattle, and the like, and aretypically non-human mammals. In a specific embodiment, a knockout mouseis produced. Knockout animals are expected to develop or be predisposedto developing diseases or disorders associated with hyper-angiogenicconditions and can be useful to screen for or test molecules for theability to decrease angiogenesis and thus treat or prevent such diseasesand disorders.

In another embodiment, transgenic animals that have incorporated andoverexpress ANG-7 genes have use as animal models of diseases anddisorders involving hypo-angiogenesis. Transgenic animals are expectedto develop or be predisposed to hypo-angiogenic conditions, or exhibitincreased resistance to diseases requiring angiogenesis, such as tumorformation. Thus, these animals can have use as animal models of suchdiseases and disorders, or the resistance to such diseases andconditions.

EXAMPLES

The following examples are offered to illustrate, but no to limit theclaimed invention.

Example 1

To identify members of the angiopoietin ligand family, a BLAST search(Altschul et al., Nucleic Acids Res. 25:3389-402 (1997)) was performedusing the Expressed Sequence Tag (EST) database from the National Centerfor Biotechnology Information (NCBI). The amino acid sequence of Ang-1was used as a probe. This search identified a human EST (AccessionNumber AA773234). The corresponding amino acid sequence of this ESTshowed significant sequence identity in the +2 reading frame to Ang-1.The probability (P) value was 4.4×10⁻²⁸, which strongly indicates thatthe identified EST encodes a fragment of a protein which belongs to thefamily of angiopoietins. Further proof that the EST encodes a fragmentof an angiopoietin protein (which was designated as “Ang-7”) wasobtained when a BLAST search of a Swissprot database was performed usingthe deduced amino acid sequence of EST AA773234). The P values obtainedfor alignments of Ang-1 and Ang-2 with the partial amino acid sequenceof Ang-7 were 3.2×10⁻³² and 2.6×10⁻³⁴, respectively.

A further search of the EST database identified another EST (AccessionNumber AA255590), which also encoded a portion of the ANG-7 cDNA. DNAsequence analysis revealed that the nucleotide sequence of EST AA255590overlaps a portion of the sequence of EST AA773234. Because EST AA773234was not readily available, the EST AA255590 nucleic acid was used as aprobe in subsequent experiments.

Example 2

To obtain the full-length cDNA clone encoding Ang-7, EST AA255590 wasused to probe a human cDNA library. Briefly, EST AA255590 is located invector pT7T3D-Pac (Pharmacia, Peapack, N.J.) between the Not I and EcoRI restriction endonuclease sites. The plasmid was linearized with therestriction endonuclease EcORI. An antisense [³²P] radioactively labeledRNA probe was generated using a Strip-EZ T3 kit (Ambion, Austin, Tex.),in accordance to the manufacturer's instructions. A Human Universal cDNALibrary (HUCL) was screened by hybridizing HUCL primary membranes(Stratagene, La Jolla, Calif., USA) with the radioactively-labeled RNAprobe. Hybridization and washing were performed as recommended by themanufacturer. (See Strip-EZ Kit Manual). After washing, the membrane wasexposed to a phosphorimager screen, scanned on a Fuji Bas-1500 scanner,and the intensity of the radioactive signals was evaluated with the TINA2.0 software (Raytest, Straubenhardt, Germany). Analysis of thehybridization profile revealed a signal at the position L04. Thecorresponding secondary array membrane was hybridized under the sameconditions. A hybridization signal was detected at position G19 on thesecondary array membrane. The individual clone L04G19 was obtained fromthe supplier. Analysis of the L04G19 clone revealed that it contained aninsert of about 2.2 kilobases (“kb”). DNA sequence analysis confirmedthat this cDNA clone contained the full length coding sequence of theANG-7 cDNA.

Example 3

DNA sequence analysis of the L04G19 cDNA revealed that the cDNA is 2,173base pairs (“bp”) long., which includes a short poly A tail. The ANG-7cDNA sequence is shown in FIG. 1 (SEQ ID NO:1). The cDNA has an openreading frame of 1,432 bp. The open reading frame encodes a polypeptideof 493 amino acid residues (SEQ ID NO:2; FIG. 2). A similarity alignmentof the amino acid sequence of Ang-7 with the sequences of humanangiopoietin-I (SEQ ID NO:3), human angiopoietin-2 (SEQ ID NO:4), humanangiopoietin-3 (SEQ ID NO:5) and human angiopoietin-4 (SEQ ID NO:6) wasconducted using the MEGALIGN™ Expert Sequence Analysis Software in theLASERGENE™ software package (DNASTAR, Madison, Wis.). Referring to FIG.3, the N-terminal and C-terminal portions of Ang-7 polypeptide containcharacteristic coiled-coil and fibrinogen-like domains, which are alsofound in other angiopoietins. The overall similarity index between theAng-7 polypeptide and Ang-1 and -2 polypeptides is 23.9% and 23.5%,respectively. Importantly, most of the amino acid residues which areconserved between the known angiopoietins are also present in the Ang-7(See FIG. 3). This amino acid sequence conservation confirms that theL04G19 cDNA clone encodes a member of the angiopoietin family.

Example 4

The tissue expression profile of the ANG-7 gene was examined. Briefly,the plasmid encoding EST AA255590 was linearized with restrictionendonuclease EcORI. An antisense [³²P]-radioactively-labeled RNA probewas generated using a Strip-EZ T3 kit (Ambion, Austin, Tex., USA), inaccordance with the manufacturer's instructions. A human multiple tissueexpression array (Clontech Laboratories, Inc., Palo Alto, Calif., USA)was hybridized with the radioactively labeled RNA probe. Hybridizationand washing were performed as recommended in the Strip-EZ Kit Manual.After washing, the membrane was exposed to a phosphorimager screen,scanned on a Fuji Bas-1500 scanner, and the intensity of the radioactivesignals was evaluated with the TINA 2.0 software (Raytest,Straubenhardt, Germany). The resulting histogram is presented on FIG. 4and demonstrates that the ANG-7 RNA is strongly expressed in hearttissues (atrium left and right, ventricle left and right), uterus,mammary gland and corpus callosum tissues. Expression of the ANG-7 RNAin these tissues, which are heavily vascularized, indicates that Ang-7polypeptide, like Ang-1 and -2 polypeptide, could play a role inangiogenesis.

Example 5

To test whether the ANG-7 cDNA is translated into Ang-7 polypeptide andto determine the molecular weight of the Ang-7 polypeptide, the ANG-7cDNA was amplified by polymerase chain reaction (“PCR”). For PCRamplification, a 5′ primer (5′ GCGAATTCACCATGAGGCCACTGTGCGT 3′ (SEQ IDNO:7)), which is complementary to the 5′ end of the ANG-7 cDNA, was usedin combination with a 3′ primer (5′ GGAAGCTTATGGAAGGTGTTGGGGTTCGG 3′(SEQ ID NO:8)), which is complementary to the 3′ end of the ANG-7 cDNA.To increase translational efficiency, a Kozak translation initiationconsensus sequence was also included in the 5′ primer. To facilitatesubsequent cloning of the PCR fragment, restriction recognitionsequences for the restriction enzymes Eco RI and Hind III wereintroduced into the 5′ and 3′ primers, respectively. The amplified cDNAwas cloned into the Eco RI and Hind III restriction sites of themammalian expression vector pcDNA3.1/Myc-His(−) (Invitrogen, Groningen,Netherlands) to create pcDNA3.1/ang7/mychis.

ANG-7 RNA transcripts were synthesized from the T7 promoter ofpcDNA3.1/ang7/mychis, according to the manufacturer's instructions. Invitro translation was conducted using a rabbit reticulocyte lysate,according to the manufacturer's instructions (Promega, Madison, Wis.,USA). The resulting proteins produced by in vitro translation werelabeled using [³⁵S]methionine. The labeled proteins were separated byelectrophoresis on a sodium dodecyl sulfate-12% polyacrylamide gel andvisualized by autoradiography. FIG. 5 depicts the results of thisexperiment. Specifically, Lane 1: Rainbow [¹⁴C]methylated proteinmolecular weight marker (Amersham, Little Chalfont Buckinghamshire,England) which included the following proteins: ovalbumin (46 kDa),carbonic anhydrase (30 kDa), trypsin inhibitor (21.5 kDa), lysozyme(14.3 kDa), and aprotinin (6.5 kDa). Lane 2 contained in vitrotranslation products of ANG-7 RNA using the T7 promoter of the mammalianexpression vector pcDNA3.1/Myc-His(−) (Invitrogen, Groningen,Netherlands). Lane 3 contained the in vitro translation products usingthe SP6 promoter of the mammalian expression vector pcDNA3.1/Myc-His(−)(negative control). Lane 4 contained a positive control from the invitro translation system (Promega, Madison, USA).

A major band of 60 kilodaltons was detected. The observed molecular massof the major band was slightly larger than the calculated molecular massof recombinant Ang-7 polypeptide (57.1 kDa). This difference may beexplained by glycosylation of several potential glycosylation sites inthe Ang-7 polypeptide.

Example 6

To determine whether ANG-7 cDNA can be expressed recombinantly in vivoin eukaryotic cells, the ANG-7 cDNA was cloned into three eukaryoticexpression vectors, and subsequently transfected into tissue culturecells.

The ANG-7 cDNA was cloned into the expression vector pcDNA3.1/Myc-His(−)(Invitrogen, Catalog No. V85520), as described above. ANG-7 cDNA wasalso cloned into pIRES (Clontech, Catalog No. 6060-1) and pZeo(Invitrogen, Catalog No. V850-01)). To facilitate detection andpurification, a polyhistidine tag was included in the C-terminal codingregion of each expression unit.

The expression constructs were transfected into Chinese Hamster Ovary(“CHO”) cells using the DOS PER liposomal transfection reagent(Boehringer Mannheim, Catalog No. 1781995). Briefly, 0.5×10⁶ CHO cellswere seeded into each well of a 6-well plate in basal medium (DMEM,Gibco/BRL, Catalog No. 41965-039); 2 mM L-Glutamine (Gibco/BRL, CatalogNo. 25030-024), Penicillin/Streptomycin (1500 IU/ml) (Gibco/BRL, CatalogNo. 15140-114), and 10% Fetal Bovine Serum (FBS, Sigma, Catalog No.F2442)). After overnight incubation at 37° C., the medium was discarded,and 500 μl of basal medium (without FBS) was mixed with 5 μg of DNA and25 μl of DOSPER were added to each well. The plates were incubated for15 min at room temperature and then an additional 520 μl well of thebasal medium (without FBS) was added. The cells were incubated for anadditional 3 hours at 37° C. After the incubation, an additional 3ml/well of basal medium was added. The next day, the medium wasdiscarded, and 3 ml/well of fresh basal was medium added. The cells wereincubated for additional two days, harvested, and then the cytoplasmicexpression of the Ang-7 polypeptide was analyzed in a Western blot usingTetra-His antibodies (Qiagen, Catalog No. 34670). The Western blot wasperformed according to the manufacturer's instructions, and containedthee lanes. Lane 1 contained the protein molecular weight markers; Lane2 contained media from CHO cells (negative control); and Lane 3contained the transiently transfected CHO cells expressing Ang-7polypeptide. The Western blot analysis is shown in FIG. 6, where theAng-7 polypeptide band is marked by an arrow. A band of about 57 kDa wasobserved in CHO cells transfected with the ANG-7 cDNA, but not in thecells transfected with the vector alone. (Compare lanes 2 and 3.) Thus,ANG-7 cDNA was transiently expressed in mammalian cells and produced apolypeptide of the expected molecular weight.

Example 7

To generate cell clones stably expressing recombinant Ang-7 polypeptide,ANG-7 cDNA clones were transfected into a human line and then stabletransfectants were selected. Briefly, a human embryonic kidney cellline, HEK293, was transfected with expression vectorspcDNA3.1/ang7/mychis or pIRES containing the ANG-7 cDNA under thecontrol of the Cytomegalovirus promoter. Transfection was performed asdescribed above in Example 6. Following transfection, the cells wereseeded into 96 well plates and cultured in selection media (Basal mediumplus 0.75 mg/ml Geneticin (Gibco/BRL, Catalog No. 10131-027)).

Recombinant expression of Ang-7 was detected by immunofluorescentstaining on Lab-Tek chamber slides (Nalgene Nunc, Catalog No. 154534)using a Tetra-His antibody (Qiagen, Catalog No. 34670) and FITC labeledsecondary antibody (Goat anti mouse IgG-FITC conjugate (Dianova, CatalogNo. 115-095-062)). About 4×10⁴ cells were seeded per well of the Lab-Tekslide and incubated overnight in a CO₂ incubator. The medium wasdiscarded, and the cells were washed once with PBS (PBS Dulbecco's w/oCalcium/Magnesium/Sodium bicarbonate (Gibco/BRL, Catalog No.14190-094)). The cells were then fixed by addition of 200 μl/well ofice-cold methanol and incubation at −20° C. for 10 min. After fixation,the cells were washed 2× with PBS containing 3% BSA (PBS-BSA), and thenblocked for 30 min by PBS-BSA at 37° C. Following blocking, 100 μl/wellof a 1:50 dilution of the Tetra-His antibody in PBS-BSA was added. Theslides were incubated for 2 hours at 37° C. After incubation with theprimary antibody, the cells were washed 10× with PBS and then 100μl/well of a 1:100 dilution of the secondary antibody (Goat anti mouseIgG-FITC conjugate) was added. The slides were incubated for 1 hour at37° C. The slides were washed 10× with PBS, the well separating grid wasremoved, and then the slides were covered with the anti-fading mountingmedium ROTI^(R)Histokit (Carl Roth, Catalog No. 6638.1) and a coverglass. After hardening of the mounting medium (usually overnight), theslides were observed in a fluorescent microscope. Positive clones wereexpanded into 24-well plates, and the expression level of recombinantprotein was compared by analyzing cell lysates on a western blot asdescribed above in Example 6. The Western blot analysis is depicted inFIG. 7. Lane 1 of the Western blot contained the protein molecularweight markers; Lane 2 contained media from HEK293 cells (negativecontrol); and Lane 3 contained stably transfected HEK293 cellsexpressing Ang-7 polypeptide. Positive clones were selected for furtheranalysis, as described below.

The Ang-7 polypeptide has a secretion signal on its N-terminus, whichsuggests that it is a secreted protein. To determine whetherrecombinantly expressed Ang-7 polypeptide is secreted by cells,conditioned media from a stably transfected HEK293 cell clone, number62, was analyzed for the presence of Ang-7 polypeptide. Briefly, theprotein was partially purified from conditioned media using a Ni-NTAagarose resin (Qiagen, # 304050), as more fully described in Example 8.The eluted column fractions were analyzed on a Western blot, asdescribed above. The Western blot analysis is depicted in FIG. 8. Lane 1of the Western blot contained conditioned media from a HEK293 cells(negative control); and Lane 2 contained conditioned media from thestably transfected HEK293 cell clone expressing Ang-7 polypeptide.

Referring to FIG. 8, conditioned medium from the stably transfectedHEK293 cell line contained a polypeptide that reacted with the Tetra-Hisantibody, while conditioned media from untransformed HEK293 cells lackedsuch a cross-reacting polypeptide. This experiment confirms that Ang-7polypeptide is a secreted into the media.

Example 8

To further characterize the Ang-7 polypeptide, it was purified fromconditioned culture media using Ni-NTA resin (Qiagen, # 304050).Briefly, conditioned media from clone 62 was collected after three days.One tablet of “complete” proteinase inhibitor (“Complete” proteinaseinhibitor cocktail tablets; Boehringer Mannheim (Roche Diagnostics)Catalog No. 1697498) was added to each 50 ml of collected media.Afterwards, imidazole (Sigma; Catalog No. I-2399) was added to a finalconcentration of 8 mM. The media was centrifuged for 10 min at 1600×g.The supernatant was transferred to fresh tubes, and 500 μl of 50% slurryof Ni-NTA agarose was added to each 50 ml of medium. The tubes weregently rocked at 4° C. for 60 min. The Ni-NTA resin was collected bycentrifugation at 1600×g for 10 min, and the supernatant was discarded.The resin was washed twice with wash buffer (50 mM NaH₂PO₄, pH 8.0; 300mM NaCl; 10 mM Imidazole) by suspending the resin in the washing bufferfollowed by centrifugation under the same conditions. After washing, theresin was again resuspended in wash buffer and transferred to a 1 mlcolumn (0.5-1 ml resin bed per column). Bound Ang-7 polypeptide waseluted in four 500 μl portions using elution buffer (50 mM NaH₂PO₄, pH8.0; 300 mM NaCl; 250 mM Imidazole). The fractions were analyzed byWestern blotting using the Tetra-His antibody, as described above. TheWestern blot analysis is depicted in FIG. 9A. The purity of the isolatedprotein was analyzed on a Coomassie-stained SDS gel, and this analysisis depicted in FIG. 9B.

Referring to FIG. 9A, Western blotting revealed a doublet of bands atabout 62-64 kDa. Comparison of the Western blot analysis with thecoomassie-stained SDS gel revealed a corresponding doublet of the samemolecular weights. (Compare FIGS. 9A and 9B). The presence of thedoublet of Ang-7 polypeptides suggests that different glycosylated formsof Ang-7 are secreted by stably transfected cells.

Example 9

The effect of ANG-7 gene expression on endothelial tube formation wasdetermined by examining HUVEC capillary like-organization in Matrigel.Adenovirus expression vectors containing ANG-7 cDNA were constructed byexcising ANG-7 cDNA from pcDNA3.1/ang7/mychis by digestion with Eco RVand Pme1. The resulting fragment was cloned into the corresponding sitesof pShuttle-CMV (He et al., Proc. Nat. Acad. Sci. USA 95:2509-14(1998)). pShuttle-CMV is an adenovirus shuttle vector in which thetransgene (i.e., ANG-7 cDNA) is under the control of the cytomegalovirus(“CMV”) promoter. This construct was transferred to an adenoviralbackbone by recombination in E. coli with the plasmid pAdEasy (“AdEZ”)(He et al., supra). The resulting recombinant was linearized with therestriction endonuclease Pac 1 and then transfected into HEK293 cells(Microbix Inc, Ontario, Canada) by standard methods. Ten dayspost-transfection (after the appearance of viral plaques), vectorparticles were harvested from the cells by multiple rounds offreeze-thawing. Particles were then used to infect 911 epithelial cells(Introgene, Leiden, Netherlands). To obtain sufficient vector formultiple experiments, three rounds of passage of viral particles tosuccessively larger cultures of 911 cells were performed. This viralstock was termed “Ad-Ang7.” This crude stock was used in the followingexperiment.

HUVEC were plated at 1.25×10⁴ cells per well at day zero in a 24 wellplate. The cells were infected with Ad-Ang7 stock, with the controlvector Ad-Ez, or with an Ad-VEGF stock (Vascular Endothelial GrowthFactor). 200 μl of each viral stock was added to the wells on day 0. Theplates were incubated in 5% CO₂ at 37° C. for 5 days. On day 6, thecells were passed to a 24 well plate coated with Matrigel at a densityof 3×10⁴ cells/well. The Matrigel coated plates were prepared asfollows: Matrigel basement membrane matrix (Becton Dickinson) was thawedon ice overnight at 4° C. Pre-cooled pipettes, pipette tips, plates andtubes where used. 0.3 μl/well of Matrigel (at 4° C.) was used to coatthe wells of a 24 well plate. The Matrigel was polymerized at 37° C. for2 hours. Following addition of HUVEC to the Matrigel coated wells, theplates was incubated for 24 hours at 5% CO₂ and 37° C. All tests wereperformed in triplicate wells. The final volume in each well is 1 ml.

After 24 hours, incubation at 37° C., 5% CO₂, each well was checkedunder a microscope at low magnification (inverted microscope at ×10power). The plates were then stained with Diff-Quick, and pictures werebe taken to compare the controls with the different concentrations.

The following tables summarize the protocol.

TABLE 1 No Matrigel Medium No Matrigel treatment (5 days) VolumeVolume 1. HUVEC Control (6 wells)  0 1 ml. 2. 911 Sup. plus cell Lysate200 μl. 800 μl. 3. Ad-EZ plus cell lysate 200 μl 800 μl 4. Ad-VEGF pluscell Lysate 200 μl. 800 μ. 5. Ad-Ang7 plus cell Lysate 200 μl. 800 μl.

TABLE 2 Matrigel Treatment Matrigel Treatment (Day 6) Volume MediumVolume 1. HUVEC Control  0  1 ml. 2. 911 Sup. plus cell lysate 200 μl.800 μl. 3. Ad-EZ plus cell lysate 200 μl 800 μl 4. Ad-VEGF plus celllysate 200 μl. 800 μl. 5. Ad-Ang7 plus cell lysate 200 μl. 800 μl.

The results were as follows, where the length of the tubing formation isgiven in centimeters. (SEM indicates standard error of the mean.)

TABLE 3 Endothelial Tubing Formation Condition Control 911 Lys. Ad-EZAd-VEGF Ad-Ang7 Well 1 68.22 82 60.8 78.6 35 Well 2 81.4 88.8 63.2 69.631.8 Well 3 70.56 75.2 89.4 68.8 36.2 Avg. 73.39 82.00 71.13 72.33 34.33SEM 4.06 4.81 9.16 3.14 1.31

As can be seen from this example, the control cells and cells infectedwith the vector alone (Ad-EZ) showed similar amounts of tubingformation. Expression of VEGF also produced similar levels of tubingformation. In contrast, expression of Ang-7 polypeptide markedlyinhibited tubing formation. Thus, this experiment demonstrates thatAng-7 polypeptide inhibits angiogenesis.

Example 10

The effect of in vivo expression of ANG-7 in B16 murine melanomametastasis was examined. Briefly, the ANG-7 cDNA was delivered ex vivowith an adenoviral vector (Ad-Ang-7) followed by introduction of thetransfected cells into mice. 74 females C 57 B1/6, 6-8 weeks old, wereused. For ex vivo administration, either crude lysates (prepared asdescribed above in Example 9) or purified vectors were used. Theadenoviral vectors were purified as follows: The protocol is amodification of Fallux et al. (Human Gene Therapy 7:215-22 (1996)). Forlarge scale purification, 911 cells were plated in a Multi-tray CellFactory (Nuclon. Denmark). When the cells reached 85% confluence, theywere infected with the recombinant adenovirus. Following infection,about 48-72 hours post-infection, when the cells showed a cytopathiceffect, the cells were harvested and centrifuged. The cell pellet wasresuspended in a small volume of medium, three cycles of freezing andthawing were performed, and the disrupted cells were pelleted to removethe cellular debris. The viral particle-containing supernatants werelayered onto a discontinuous cesium chloride (CsCl) gradient composed of1 ml at d=1.4 g/ml overlaid with 3 mls of d=1.25 g/ml. The gradientswere centrifuged at 151,000×g for 2 hours. The opaque band of virusparticles, at the 1.25/1.4 density boundary, was collected and loadedonto a homogeneous CsCl solution of d=1.3 g/ml. This second gradient wasspun at 151,000×g for 18 hours. The single band of virus particles wascollected and dialyzed twice for one hour against 0.135M NaCl, 1 mMMgCl₂, 10 mM Tris pH 7.5. The second and final dialysis was carried outagainst the same buffer with the addition of 10% glycerol. Stock titerswere determined by plaque assay using 293 or 911 cells.

B16.F10 cells, from a murine melanoma metastasis were infected for 24hours with one of the following adenovirus expression vectors: Ad-E1(control), Ad-Ang7 or Ad-VEGF. The infected cells were injectedintravenously into the lateral tail vein of the mice at the end of the24 incubation period. The cell concentration of each injection was 2×10⁵cells in 0.2 ml of PBS. Day 0 was the date of injection into mice.

The animals were weighed two times a week. Two animals from group 1(control) were sacrificed on day 14, the lungs collected and the numberof metastases determined. Following counting of the metastases, theremainder of the animals (10 in each group) were sacrificed on day 14.At that time, lungs were collected, weighed and the number of metastasescounted.

The following table summarizes the experimental protocol.

TABLE 4 NUMBER OF GROUP ANIMALS CELL INFECTION SACRIFICED ON 1 10 NoneDay 14 (only 2 mice), 2 10 AdEZ Day 14 6 9 Ad-VEGF Day 14 8 9 Ad-Ang7Day 14

The following Table 5 summarizes the lung weights from each group. SEMis the “standard error of the mean.”

TABLE 5 Lung Weight Group Average SEM Median 1 (control) 200.4 63.4203.2 2 (AdEz) 219.0 69.3 222.5 6 (Ad-VEGF) 173.3 61.3 172.6 8 (Ad-Ang7)168.6 56.2 168.6

The following Table 6 summarizes the number of lung metastases from eachgroup. SEM is the “standard error of the mean.”

TABLE 6 Lung Metastases Group Average SEM Median 1 (control) 50.9 12.339.5 2 (AdEZ) 24.3 7.1 27.7 6 (Ad-VEGF) 48.3 10.0 47.1 8 (Ad-Ang7) 3.02.1 0.0

As can be seen from this data, the average lung weight in mice receivingtumor cells overexpressing ANG-7 cDNA was markedly lower than in controlanimals. The average lung weight in AD-VEGF and Ad-Ang7 treated animalswas similar. More importantly, and referring to Table 6, the averagenumber of lung metastases was over 10 times less in tumor cellsoverexpressing Ang-7 polypeptide as compared with control andVEGF-treated animals. Thus, this data reveal that overexpression ofANG-7 cDNA inhibits tumor cell growth.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

8 1 2173 DNA Homo sapiens 1 gaaaatgagg ctgctgcgga cggcctgagg atgaaccccaagccctggac ctgccgagcg 60 tggcactgag gcagcggctg acgctactgt gagggaaagaaggttgtgag cagccccgca 120 ggacccctgg ccagccctgg ccccagcctc tgccggagccctctgtggag gcagagccag 180 tggagcccag tgaggcaggg ctgcttggca gccaccggcctgcaactcag gaacccctcc 240 agaggccatg gacaggctgc cccgctgacg gccagggtgaagcatgtgag gagccgcccc 300 ggagccaagc aggagggaag aggctttcat agattctattcacaaagaat aaccaccatt 360 ttgcaaagac catgaggcca ctgtgcgtga catgctggtggctcggactg ctggctgcca 420 tgggagctgt tgcaggccag gaggacggtt ttgagggcactgaggagggc tcgccaagag 480 agttcattta cctaaacagg tacaagcggg cgggcgagtcccaggacaag tgcacctaca 540 ccttcattgt gccccagcag cgggtcacgg gtgccatctgcgtcaactcc aaggagcctg 600 aggtgcttct ggagaaccga gtgcataagc aggagctagagctgctcaac aatgagctgc 660 tcaagcagaa gcggcagatc gagacgctgc agcagctggtgaaggtggac ggcggcattg 720 tgagcgaggt gaagctgctg cgcaaggaga gccgcaacatgaactcgcgg gtcacgcagc 780 tctacatgca gctcctgcac gagatcatcc gcaagcgggacaacgcgttg gagctctccc 840 agctggagaa caggatcctg aaccagacag ccgacatgctgcagctggcc agcaagtaca 900 aggacctgga gcacaagtac cagcacctgg ccacactggcccacaaccaa tcagagatca 960 tcgcgcagct tgaggagcac tgccagaggg tgccctcggccaggcccgtc ccccagccac 1020 cccccgctgc cccgccccgg gtctaccaac cacccacctacaaccgcatc atcaaccaga 1080 tctctaccaa cgagatccag agtgaccaga acctgaaggtgctgccaccc cctctgccca 1140 ctatgcccac tctcaccagc ctcccatctt ccaccgacaagccgtcgggc ccatggagag 1200 actgcctgca ggccctggag gatggccacg acaccagctccatctacctg gtgaagccgg 1260 agaacaccaa ccgcctcatg caggtgtggt gcgaccagagacacgacccc gggggctgga 1320 ccgtcatcca gagacgcctg gatggctctg ttaacttcttcaggaactgg gagacgtaca 1380 agcaagggtt tgggaacatt gacggcgaat actggctgggcctggagaac atttactggc 1440 tgacgaacca aggcaactac aaactcctgg tgaccatggaggactggtcc ggccgcaaag 1500 tctttgcaga atacgccagt ttccgcctgg aacctgagagcgagtattat aagctgcggc 1560 tggggcgcta ccatggcaat gcgggtgact cctttacatggcacaacggc aagcagttca 1620 ccaccctgga cagagatcat gatgtctaca caggaaactgtgcccactac cagaagggag 1680 gctggtggta taacgcctgt gcccactcca acctcaacggggtctggtac cgcgggggcc 1740 attaccggag ccgctaccag gacggagtct actgggctgagttccgagga ggctcttact 1800 cactcaagaa agtggtgatg atgatccgac cgaaccccaacaccttccac taagccagct 1860 ccccctcctg acctctcgtg gccattgcca ggagcccaccctggtcacgc tggccacagc 1920 acaaagaaca actcctcacc agttcatcct gaggctgggaggaccgggat gctggattct 1980 gttttccgaa gtcactgcag cggatgatgg aactgaatcgatacggtgtt ttctgtccct 2040 cctactttcc ttcacaccag acagcccctc atgtctccaggacaggacag gactacagac 2100 aactctttct ttaaataaat taagtctcta caataaaaacacaactgcaa agtaaaaaaa 2160 aaaaaaaaaa aaa 2173 2 493 PRT Homo sapiens 2Met Arg Pro Leu Cys Val Thr Cys Trp Trp Leu Gly Leu Leu Ala Ala 1 5 1015 Met Gly Ala Val Ala Gly Gln Glu Asp Gly Phe Glu Gly Thr Glu Glu 20 2530 Gly Ser Pro Arg Glu Phe Ile Tyr Leu Asn Arg Tyr Lys Arg Ala Gly 35 4045 Glu Ser Gln Asp Lys Cys Thr Tyr Thr Phe Ile Val Pro Gln Gln Arg 50 5560 Val Thr Gly Ala Ile Cys Val Asn Ser Lys Glu Pro Glu Val Leu Leu 65 7075 80 Glu Asn Arg Val His Lys Gln Glu Leu Glu Leu Leu Asn Asn Glu Leu 8590 95 Leu Lys Gln Lys Arg Gln Ile Glu Thr Leu Gln Gln Leu Val Lys Val100 105 110 Asp Gly Gly Ile Val Ser Glu Val Lys Leu Leu Arg Lys Glu SerArg 115 120 125 Asn Met Asn Ser Arg Val Thr Gln Leu Tyr Met Gln Leu LeuHis Glu 130 135 140 Ile Ile Arg Lys Arg Asp Asn Ala Leu Glu Leu Ser GlnLeu Glu Asn 145 150 155 160 Arg Ile Leu Asn Gln Thr Ala Asp Met Leu GlnLeu Ala Ser Lys Tyr 165 170 175 Lys Asp Leu Glu His Lys Tyr Gln His LeuAla Thr Leu Ala His Asn 180 185 190 Gln Ser Glu Ile Ile Ala Gln Leu GluGlu His Cys Gln Arg Val Pro 195 200 205 Ser Ala Arg Pro Val Pro Gln ProPro Pro Ala Ala Pro Pro Arg Val 210 215 220 Tyr Gln Pro Pro Thr Tyr AsnArg Ile Ile Asn Gln Ile Ser Thr Asn 225 230 235 240 Glu Ile Gln Ser AspGln Asn Leu Lys Val Leu Pro Pro Pro Leu Pro 245 250 255 Thr Met Pro ThrLeu Thr Ser Leu Pro Ser Ser Thr Asp Lys Pro Ser 260 265 270 Gly Pro TrpArg Asp Cys Leu Gln Ala Leu Glu Asp Gly His Asp Thr 275 280 285 Ser SerIle Tyr Leu Val Lys Pro Glu Asn Thr Asn Arg Leu Met Gln 290 295 300 ValTrp Cys Asp Gln Arg His Asp Pro Gly Gly Trp Thr Val Ile Gln 305 310 315320 Arg Arg Leu Asp Gly Ser Val Asn Phe Phe Arg Asn Trp Glu Thr Tyr 325330 335 Lys Gln Gly Phe Gly Asn Ile Asp Gly Glu Tyr Trp Leu Gly Leu Glu340 345 350 Asn Ile Tyr Trp Leu Thr Asn Gln Gly Asn Tyr Lys Leu Leu ValThr 355 360 365 Met Glu Asp Trp Ser Gly Arg Lys Val Phe Ala Glu Tyr AlaSer Phe 370 375 380 Arg Leu Glu Pro Glu Ser Glu Tyr Tyr Lys Leu Arg LeuGly Arg Tyr 385 390 395 400 His Gly Asn Ala Gly Asp Ser Phe Thr Trp HisAsn Gly Lys Gln Phe 405 410 415 Thr Thr Leu Asp Arg Asp His Asp Val TyrThr Gly Asn Cys Ala His 420 425 430 Tyr Gln Lys Gly Gly Trp Trp Tyr AsnAla Cys Ala His Ser Asn Leu 435 440 445 Asn Gly Val Trp Tyr Arg Gly GlyHis Tyr Arg Ser Arg Tyr Gln Asp 450 455 460 Gly Val Tyr Trp Ala Glu PheArg Gly Gly Ser Tyr Ser Leu Lys Lys 465 470 475 480 Val Val Met Met IleArg Pro Asn Pro Asn Thr Phe His 485 490 3 498 PRT Homo sapiens 3 Met ThrVal Phe Leu Ser Phe Ala Phe Leu Ala Ala Ile Leu Thr His 1 5 10 15 IleGly Cys Ser Asn Gln Arg Arg Ser Pro Glu Asn Ser Gly Arg Arg 20 25 30 TyrAsn Arg Ile Gln His Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro 35 40 45 GluHis Asp Gly Asn Cys Arg Glu Ser Thr Thr Asp Gln Tyr Asn Thr 50 55 60 AsnAla Leu Gln Arg Asp Ala Pro His Val Glu Pro Asp Phe Ser Ser 65 70 75 80Gln Lys Leu Gln His Leu Glu His Val Met Glu Asn Tyr Thr Gln Trp 85 90 95Leu Gln Lys Leu Glu Asn Tyr Ile Val Glu Asn Met Lys Ser Glu Met 100 105110 Ala Gln Ile Gln Gln Asn Ala Val Gln Asn His Thr Ala Thr Met Leu 115120 125 Glu Ile Gly Thr Ser Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys130 135 140 Leu Thr Asp Val Glu Thr Gln Val Leu Asn Gln Thr Ser Arg LeuGlu 145 150 155 160 Ile Gln Leu Leu Glu Asn Ser Leu Ser Thr Tyr Lys LeuGlu Lys Gln 165 170 175 Leu Leu Gln Gln Thr Asn Glu Ile Leu Lys Ile HisGlu Lys Asn Ser 180 185 190 Leu Leu Glu His Lys Ile Leu Glu Met Glu GlyLys His Lys Glu Glu 195 200 205 Leu Asp Thr Leu Lys Glu Glu Lys Glu AsnLeu Gln Gly Leu Val Thr 210 215 220 Arg Gln Thr Tyr Ile Ile Gln Glu LeuGlu Lys Gln Leu Asn Arg Ala 225 230 235 240 Thr Thr Asn Asn Ser Val LeuGln Lys Gln Gln Leu Glu Leu Met Asp 245 250 255 Thr Val His Asn Leu ValAsn Leu Cys Thr Lys Glu Gly Val Leu Leu 260 265 270 Lys Gly Gly Lys ArgGlu Glu Glu Lys Pro Phe Arg Asp Cys Ala Asp 275 280 285 Val Tyr Gln AlaGly Phe Asn Lys Ser Gly Ile Tyr Thr Ile Tyr Ile 290 295 300 Asn Asn MetPro Glu Pro Lys Lys Val Phe Cys Asn Met Asp Val Asn 305 310 315 320 GlyGly Gly Trp Thr Val Ile Gln His Arg Glu Asp Gly Ser Leu Asp 325 330 335Phe Gln Arg Gly Trp Lys Glu Tyr Lys Met Gly Phe Gly Asn Pro Ser 340 345350 Gly Glu Tyr Trp Leu Gly Asn Glu Phe Ile Phe Ala Ile Thr Ser Gln 355360 365 Arg Gln Tyr Met Leu Arg Ile Glu Leu Met Asp Trp Glu Gly Asn Arg370 375 380 Ala Tyr Ser Gln Tyr Asp Arg Phe His Ile Gly Asn Glu Lys GlnAsn 385 390 395 400 Tyr Arg Leu Tyr Leu Lys Gly His Thr Gly Thr Ala GlyLys Gln Ser 405 410 415 Ser Leu Ile Leu His Gly Ala Asp Phe Ser Thr LysAsp Ala Asp Asn 420 425 430 Asp Asn Cys Met Cys Lys Cys Ala Leu Met LeuThr Gly Gly Trp Trp 435 440 445 Phe Asp Ala Cys Gly Pro Ser Asn Leu AsnGly Met Phe Tyr Thr Ala 450 455 460 Gly Gln Asn His Gly Lys Leu Asn GlyIle Lys Trp His Tyr Phe Lys 465 470 475 480 Gly Pro Ser Tyr Ser Leu ArgSer Thr Thr Met Met Ile Arg Pro Leu 485 490 495 Asp Phe 4 496 PRT Homosapiens 4 Met Trp Gln Ile Val Phe Phe Thr Leu Ser Cys Asp Leu Val LeuAla 1 5 10 15 Ala Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser Ile GlyLys Lys 20 25 30 Gln Tyr Gln Val Gln His Gly Ser Cys Ser Tyr Thr Phe LeuLeu Pro 35 40 45 Glu Met Asp Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val SerAsn Ala 50 55 60 Val Gln Arg Asp Ala Pro Leu Glu Tyr Asp Asp Ser Val GlnArg Leu 65 70 75 80 Gln Val Leu Glu Asn Ile Met Glu Asn Asn Thr Gln TrpLeu Met Lys 85 90 95 Leu Glu Asn Tyr Ile Gln Asp Asn Met Lys Lys Glu MetVal Glu Ile 100 105 110 Gln Gln Asn Ala Val Gln Asn Gln Thr Ala Val MetIle Glu Ile Gly 115 120 125 Thr Asn Leu Leu Asn Gln Thr Ala Glu Gln ThrArg Lys Leu Thr Asp 130 135 140 Val Glu Ala Gln Val Leu Asn Gln Thr ThrArg Leu Glu Leu Gln Leu 145 150 155 160 Leu Glu His Ser Leu Ser Thr AsnLys Leu Glu Lys Gln Ile Leu Asp 165 170 175 Gln Thr Ser Glu Ile Asn LysLeu Gln Asp Lys Asn Ser Phe Leu Glu 180 185 190 Lys Lys Val Leu Ala MetGlu Asp Lys His Ile Ile Gln Leu Gln Ser 195 200 205 Ile Lys Glu Glu LysAsp Gln Leu Gln Val Leu Val Ser Lys Gln Asn 210 215 220 Ser Ile Ile GluGlu Leu Glu Lys Lys Ile Val Thr Ala Thr Val Asn 225 230 235 240 Asn SerVal Leu Gln Lys Gln Gln His Asp Leu Met Glu Thr Val Asn 245 250 255 AsnLeu Leu Thr Met Met Ser Thr Ser Asn Ser Ala Lys Asp Pro Thr 260 265 270Val Ala Lys Glu Glu Gln Ile Ser Phe Arg Asp Cys Ala Glu Val Phe 275 280285 Lys Ser Gly His Thr Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro Asn 290295 300 Ser Thr Glu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly Gly Gly305 310 315 320 Gly Trp Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val AspPhe Gln 325 330 335 Arg Thr Trp Lys Glu Tyr Lys Val Gly Phe Gly Asn ProSer Gly Glu 340 345 350 Tyr Trp Leu Gly Asn Glu Phe Val Ser Gln Leu ThrAsn Gln Gln Arg 355 360 365 Tyr Val Leu Lys Ile His Leu Lys Asp Trp GluGly Asn Glu Ala Tyr 370 375 380 Ser Leu Tyr Glu His Phe Tyr Leu Ser SerGlu Glu Leu Asn Tyr Arg 385 390 395 400 Ile His Leu Lys Gly Leu Thr GlyThr Ala Gly Lys Ile Ser Ser Ile 405 410 415 Ser Gln Pro Gly Asn Asp PheSer Thr Lys Asp Gly Asp Asn Asp Lys 420 425 430 Cys Ile Cys Lys Cys SerGln Met Leu Thr Gly Gly Trp Trp Phe Asp 435 440 445 Ala Cys Gly Pro SerAsn Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln 450 455 460 Asn Thr Asn LysPhe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys Gly Ser 465 470 475 480 Gly TyrSer Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe 485 490 495 5509 PRT Homo sapiens 5 Met Leu Cys Gln Pro Ala Met Leu Leu Asp Gly LeuLeu Leu Leu Ala 1 5 10 15 Thr Met Ala Ala Ala Gln His Arg Gly Pro GluAla Gly Gly His Arg 20 25 30 Gln Ile His Gln Val Arg Arg Gly Gln Cys SerTyr Thr Phe Val Val 35 40 45 Pro Glu Pro Asp Ile Cys Gln Leu Ala Pro ThrAla Ala Pro Glu Ala 50 55 60 Leu Gly Gly Ser Asn Ser Leu Gln Arg Asp LeuPro Ala Ser Arg Leu 65 70 75 80 His Leu Thr Asp Trp Arg Ala Gln Arg AlaGln Arg Ala Gln Arg Val 85 90 95 Ser Gln Leu Glu Lys Ile Leu Glu Asn AsnThr Gln Trp Leu Leu Lys 100 105 110 Leu Glu Gln Ser Ile Lys Val Asn LeuArg Ser His Leu Val Gln Ala 115 120 125 Gln Gln Asp Thr Ile Gln Asn GlnThr Thr Thr Met Leu Ala Leu Gly 130 135 140 Ala Asn Leu Met Asn Gln ThrLys Ala Gln Thr His Lys Leu Thr Ala 145 150 155 160 Val Glu Ala Gln ValLeu Asn Gln Thr Leu His Met Lys Thr Gln Met 165 170 175 Leu Glu Asn SerLeu Ser Thr Asn Lys Leu Glu Arg Gln Met Leu Met 180 185 190 Gln Ser ArgGlu Leu Gln Arg Leu Gln Gly Arg Asn Arg Ala Leu Glu 195 200 205 Thr ArgLeu Gln Ala Leu Glu Ala Gln His Gln Ala Gln Leu Asn Ser 210 215 220 LeuGln Glu Lys Arg Glu Gln Leu His Ser Leu Leu Asp His Gln Thr 225 230 235240 Gly Thr Leu Ala Asn Leu Lys His Asn Leu His Ala Leu Ser Ser Asn 245250 255 Ser Ser Ser Leu Gln Gln Gln Gln Gln Gln Leu Thr Glu Phe Val Gln260 265 270 Arg Leu Val Arg Ile Val Ala Gln Asp Gln His Pro Val Ser LeuLys 275 280 285 Thr Pro Lys Pro Val Phe Gln Asp Cys Ala Glu Ile Lys ArgSer Gly 290 295 300 Val Asn Thr Ser Gly Val Tyr Thr Ile Tyr Glu Thr AsnMet Thr Lys 305 310 315 320 Pro Leu Lys Val Phe Cys Asp Met Glu Thr AspGly Gly Gly Trp Thr 325 330 335 Leu Ile Gln His Arg Glu Asp Gly Ser ValAsn Phe Gln Arg Thr Trp 340 345 350 Glu Glu Tyr Lys Glu Gly Phe Gly AsnVal Ala Arg Glu His Trp Leu 355 360 365 Gly Asn Glu Ala Val His Arg LeuThr Ser Arg Thr Ala Tyr Leu Leu 370 375 380 Arg Val Glu Leu His Asp TrpGlu Gly Arg Gln Thr Ser Ile Gln Tyr 385 390 395 400 Glu Asn Phe Gln LeuGly Ser Glu Arg Gln Arg Tyr Ser Leu Ser Val 405 410 415 Asn Asp Ser SerSer Ser Ala Gly Arg Lys Asn Ser Leu Ala Pro Gln 420 425 430 Gly Thr LysPhe Ser Thr Lys Asp Met Asp Asn Asp Asn Cys Met Cys 435 440 445 Lys CysAla Gln Met Leu Ser Gly Gly Trp Trp Phe Asp Ala Cys Gly 450 455 460 LeuSer Asn Leu Asn Gly Ile Tyr Tyr Ser Val His Gln His Leu His 465 470 475480 Lys Ile Asn Gly Ile Arg Trp His Tyr Phe Arg Gly Pro Ser Tyr Ser 485490 495 Leu His Gly Thr Arg Met Met Leu Arg Pro Met Gly Ala 500 505 6504 PRT Homo sapiens 6 Asn Leu Ser Gln Leu Ala Met Leu Gln Gly Ser LeuLeu Leu Val Val 1 5 10 15 Ala Thr Met Ser Val Ala Gln Gln Thr Arg GlnGlu Ala Asp Arg Gly 20 25 30 Cys Glu Thr Leu Val Val Gln His Gly His CysSer Tyr Thr Phe Leu 35 40 45 Leu Pro Lys Ser Glu Pro Cys Pro Pro Gly ProGlu Val Ser Arg Asp 50 55 60 Ser Asn Thr Leu Gln Arg Glu Ser Leu Ala AsnPro Leu His Leu Gly 65 70 75 80 Lys Leu Pro Thr Gln Gln Val Lys Gln LeuGlu Gln Ala Leu Gln Asn 85 90 95 Asn Thr Gln Val Leu Lys Lys Leu Glu ArgAla Ile Lys Thr Ile Leu 100 105 110 Arg Ser Lys Leu Glu Gln Val Gln GlnGln Met Ala Gln Asn Gln Thr 115 120 125 Ala Pro Met Leu Glu Leu Gly ThrSer Leu Leu Asn Gln Thr Thr Ala 130 135 140 Gln Ile Arg Lys Leu Thr AspMet Glu Ala Gln Leu Leu Asn Gln Thr 145 150 155 160 Ser Arg Met Asp AlaGln Met Pro Glu Thr Phe Leu Ser Thr Asn Lys 165 170 175 Leu Glu Asn GlnLeu Leu Leu Gln Arg Gln Lys Leu Gln Gln Leu Gln 180 185 190 Gly Gln AsnSer Ala Leu Glu Lys Arg Leu Gln Ala Leu Glu Thr Lys 195 200 205 Gln GlnGlu Glu Leu Ala Ser Glu Leu Ser Lys Lys Ala Lys Leu Leu 210 215 220 AsnThr Leu Ser Arg Gln Ser Ala Ala Leu Thr Asn Glu Glu Arg Gly 225 230 235240 Leu Arg Gly Val Arg His Asn Ser Ser Leu Leu Gln Asp Gln Gln His 245250 255 Ser Leu Arg Gln Leu Leu Val Leu Leu Arg His Leu Val Gln Glu Arg260 265 270 Ala Asn Ala Ser Ala Pro Ala Phe Ile Met Ala Gly Glu Gln ValPhe 275 280 285 Gln Asp Cys Ala Glu Ile Gln Arg Ser Gly Ala Ser Ala SerGly Phe 290 295 300 Tyr Thr Ile Gln Val Ser Asn Ala Thr Lys Pro Arg LysVal Phe Cys 305 310 315 320 Asp Leu Gln Ser Ser Gly Gly Arg Val Thr LeuIle Gln Arg Arg Glu 325 330 335 Asn Gly Thr Val Asn Phe Gln Arg Asn ValLys Asp Tyr Lys Gln Gly 340 345 350 Phe Gly Asp Pro Ala Gly Glu His ValGlu Leu Gly Asn Glu Val Val 355 360 365 His Gln Leu Thr Arg Arg Ala AlaTyr Ser Leu Arg Val Glu Leu Gln 370 375 380 Asp Val Glu Gly His Glu AlaTyr Ala Gln Tyr Glu His Phe His Leu 385 390 395 400 Gly Ser Glu Asn GlnLeu Tyr Arg Leu Ser Val Val Gly Tyr Ser Gly 405 410 415 Ser Ala Gly ArgGln Ser Ser Leu Val Leu Gln Asn Thr Ser Phe Ser 420 425 430 Thr Leu AspSer Asp Asn Asp His Cys Leu Cys Lys Cys Ala Gln Val 435 440 445 Met SerGly Gly Trp Trp Phe Asp Ala Cys Gly Leu Ser Asn Leu Asn 450 455 460 AspVal Tyr Tyr His Ala Pro Asp Asn Lys Tyr Lys Met Asp Gly Glu 465 470 475480 Arg Val His Tyr Phe Lys Gly Pro Ser Tyr Ser Leu Arg Ala Ser Arg 485490 495 Met Met Glu Arg Pro Leu Asp Glu 500 7 28 DNA Homo sapiens 7gcgaattcac catgaggcca ctgtgcgt 28 8 29 DNA Homo sapiens 8 ggaagcttatggaaggtgtt ggggttcgg 29

What is claimed is:
 1. A method for inhibiting angiogenesis comprisingadministering to a mammal a therapeutically effective dose of anisolated Ang-7 polypeptide, wherein said polypeptide comprises SEQ IDNO:2.
 2. The method of claim 1, wherein the mammal is human.
 3. Themethod of claim 1, wherein the administering is performed in vivo. 4.The method of claim 1, further comprising administering a pharmaceuticalcarrier.
 5. The method of claim 1, wherein the administering is ex vivo.6. The method of claim 1, wherein the Ang-7 polypeptide is recombinantlyexpressed.
 7. The method of claim 6, wherein the Ang-7 polypeptide isrecombinantly expressed by culturing a cell containing an ANG-7 nucleicacid under conditions which result in expression of the polypeptide, andrecovering the Ang-7 polypeptide from the cell culture.
 8. The method ofclaim 6, wherein the Ang-7 polypeptide is expressed in E. coli.
 9. Themethod of claim 6, wherein the Ang-7 polypeptide is expressed inmammalian cells.
 10. The method of claim 7, wherein the ANG-7 nucleicacid is operably linked to a promoter in an expression vector.
 11. Themethod of claim 7, wherein the ANG-7 nucleic acid has the sequence ofhuman ANG-7 (SEQ ID NO:1).
 12. A method of treating a tumor in a subjectin need of anti-angiogenesis therapy comprising administering to asubject a therapeutically effective amount of an Ang-7 polypeptide,wherein said polypeptide comprises SEQ ID NO:2.
 13. The method of claim12, wherein the subject is human.
 14. The method of claim 12, whereinthe administering is performed in vivo.
 15. The method of claim 12,further comprising administering a pharmaceutical carrier.
 16. Themethod of claim 12, wherein the administration is ex vivo.
 17. Themethod of claim 12, wherein the Ang-7 polypeptide is recombinantlyexpressed.
 18. The method of claim 17, wherein the Ang-7 polypeptide isrecombinantly expressed by culturing a cell containing an ANG-7 nucleicacid under conditions which result in expression of the polypeptide, andrecovering the Ang-7 polypeptide from the cell culture.
 19. The methodof claim 17, wherein the Ang-7 polypeptide is expressed in mammaliancells, yeast cells, bacterial cells, or insect cells.
 20. The method ofclaim 17, wherein the Ang-7 polypeptide is expressed in mammalian cells.21. The method of claim 18, wherein the ANG-7 nucleic acid is operablylinked to a promoter in an expression vector.
 22. The method of claim18, wherein the expression vector is an adenoviral vector, a retroviralvector, or a lentiviral vector.
 23. The method of claim 18, wherein theANG-7 nucleic acid has the sequence of human ANG-7 (SEQ ID NO:1).
 24. Amethod of inhibiting endothelial tube formation comprising administeringto an endothelial cell an effective amount of an Ang-7 polypeptide,wherein said polypeptide comprises SEQ ID NO:2.
 25. A method ofinhibiting tumor cell growth comprising administering to a tumor cell aneffect amount of an Ang-7 polypeptide, wherein said polypeptidecomprises SEQ ID NO:2.
 26. A method of inhibiting tumor cell growthcomprising administering to a tumor cell an effective amount of an Ang-7polypeptide, or fragment, variant, derivative, analog or apharmaceutically acceptable salt thereof.
 27. The method of claim 26,wherein the Ang-7 polypeptide is human Ang-7.